Kristin Doan Notebook

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Latest revision as of 21:15, 15 August 2007

My Construction Files
My Sequencing Files

6/6/07

Today was a pretty easy day. We began with trying to continue the project that was very much unsuccessful yesterday. We wanted to make a composite part with the RFP insert downstream of the p-Tet promoter. We digested pBca9145-1144 to retrieve our RFP insert as well as pBca9145-9205, which contains the promoter. low and behold, we failed again. all the lanes with the RFP showed up beautifully, but no lanes showed up for the 9205 plasmid. BUT!! it may not be our fault because the 9205 DNA was never tested. so instead, we worked on our wikis. The day wasn't in total vain because I did read two articles on heme. Kdoan 17:37, 6 June 2007 (EDT)

6/7/07

We began by setting up PCRs with the new oligos we received. but there were only one set of pipetmans. since we're a team, we made do. I was overwhelmed by six PCR reaction tubes. One for hemB, one for hemD, and two each for hemA from R. Capsulatus and hemC since they both have internal restriction sites. After they completed, I ran the two PCRs for hemA and two PCRs for hemC on a gel to gel purify. Horrible result: only one PCR reaction from hemA and one PCR reaction from hemC worked. The ones that worked were the ones with oligos kd003/kd002 and kd009/kd008. As a result, I extracted/dissolved the gels of the PCR reactions that worked and dissolved in ADB to store in the fridge. I can't proceed with two good PCR products without the other two. I then set up new reactions to redo my failed PCRs. One for the failed hemA with oligos kd001/kd004 and one for the failed hemC with oligos kd007kd010 and ran these PCRs on the machines upstairs. Inbetween all of this, I ran an analytical gel to see my PCRs from hemB and hemD. Good news: they worked! I saw bands corresponding to 1006bp of hemB and another to 772bp of hemD. I then took a picture of this gel and cleaned up the PCR product. Afterwards, I digested with Bg1II/XhoI/DpnI, ligated with the vector pBca9145-1144#5 that Austin has prepared and digested for us, and then transformed into DH110B competent cells. I made one plate for hemB and another for hemD. Quite a busy series of events! Kdoan 19:20, 8 June 2007 (EDT) to do

6/8/07

2nd day: things were in a little more control today. There were colonies for both plates, white ones! I gave them more time to grow. I set up two PCR tubes for hemA from CFT073 E.Coli. Two again because there's an internal restriction site. One had oligos kd013/kd016 and another had kd015/kd014. I ran those and during this time, I received my PCRs that I had left upstairs from the day before. The PCRs that I had to redo. I ran them on a gel to gel purify and this time there were bands. I excised a 639bp band from hemA R. capsulatus and a 306kb band from hemC and dissolved in ADB buffer. I retrieved the two PCR reactions for hemA and hemC that I had refrigerated the day before and combined the two hemA products with the two hemC products. I cleaned up the gel and isolated the DNA and eluted with 15uL of water. I then ran one PCR reaction to ligate the two PCR products of hemA with kd001/kd002 and another to ligate hemC with kd007/kd008. After I ran this PCR and it completed I refrigerated the products to test them out on Monday. When the colonies of my plates had grown to adequate size, I selected four from each that I would run colony PCRs on. I labeled the ones from hemB B1-B4 and the ones from hemD D1-D4. I prepared master solutions to perform four PCRs for hemB and four for hemD. I used oligos kd005/kd006 for hemB and kd011/kd012 for hemD again. I used pipette tips to extract cells and add as a template to each PCR tube. Afterwards, I would make an "X" of cells on a fresh plate. I labeled the colonies I picked 1-4, the PCRs 1-4, and my new plate had 1-4 in each corner. I then ran a PCR with the eight rxn tubes and incubated my new hemB and hemD plates in the 37C. Austin will check on them tomorrow and move the plates to the 4C fridge. Before I left, I ran a test gel to see if my two PCR reactions for hemA from CFT073 worked. And they didnt! I'll have to set those up again on Monday. Kdoan 17:16, 11 June 2007 (EDT)

6/11/07

I started off today with running analytical gels to check if my PCRs from Friday worked. I ran one gel to see if my eight colony PCRs worked. Bad news: no bands appeared! However, I highly doubt that my colonies do not have the insert because none of them turned red. Farnazz and Amin said that their colony PCRs also often fail so I decided to move on and grow my four colonies with hemB and four colonies with hemD in culture anyway. I scraped colonies from the new plates with the "X" of cells and placed them in the shaker to incubate overnight. I ran another test gel to see my results for the other PCR reactions. The one PCR to combine the hemA from R.cap and the other PCR to combine hemC. This PCR also failed. So I set up two more PCR reactions. One to combine the hemA from R.cap and another to combine hemC and ran these upstairs. I then poured my agarose gels. Then I set up two more PCRs to amplify the hemA from CFT073. When I ran these gels, all my PCRs had failed. This was a bad day! I'll have to see what I'm doing wrong... Kdoan 17:29, 11 June 2007 (EDT)

6/12/07

So I realized that I've been adding 1ul of PCR buffer instead of 5ul. This is not good because without the buffer, the DNA polymerase degrades and nothing gets amplified. So I set up yet another PCR reaction to combine the two products of hemA and two products of hemC. While I waited, I obtained the eight cultures I grew overnight and miniprepped the four cultures of hemB and four of hemD. In order to see if my plasmids are correct and if they could possibly contain the right gene I ran test digests. I digested 5uL from each miniprep with Bg1II and Xho1 then ran all eight onto a gel. Good thing: for each lane, I observed one large band corresponding to the plasmid and a smaller band, which is most likely the gene. I looked at the sizes and the largest band was approximately 2000bp, which is the size of the plasmid. The smaller band for the hemB minipreps was around 1000bp, which is consistent and the smaller band for hemD was around 800bp, which is also good. I then picked two samples from each gene: B2, B4, D2, and D4. I sent kd001hemB2, kd002hemB4, kd003hemD2, kd004hemD4 for sequencing at Quintara. I checked my PCRs for hemA and hemC and this time I got bands! So I cleaned up the PCR products, digested with Bg1II/Xho1/Dpn1, cleaned up the digest, ligated with the vector pBca9145-1144#5, and then transformed into DH110B competent cells. I made one plate for hemA and another for hemC. Kdoan 17:50, 12 June 2007 (EDT)

6/13/07

I set up more PCRs to redo my hemA from CFT073. This hemA also has an internal restriction site so I had to divide into two PCRs. However, my CFT073 had run out! So I tried my best by adding 1uL of water and centrifuging hoping to collect any CFT073 on the sides. When I ran the PCR results on a gel, only a band showed up for the kd013/kd016 and not the kd015/kd014. So I gel extracted and dissolved the band I did see in ADB buffer and refrigerated until I get more CFT073. I then looked at my hemA and hemC plates and chose three colonies from each (hemA1-hemA-3 and hemC1-hemC3) and grew them in culture. I then analyzed my sequencing results for B2, B4, D2, and D4. D2 was short so I immediately threw that out. The first 600-650bp of B4 and D4 were good but the sequences of hemB and hemD are 1006bp and 772bp, respectively. So I sent in another 8uL of my minipreps and included G01001, the reverse primer, to get the end of my gene so I could analyze the last few hundred bp and check for mutations. Kdoan 18:26, 14 June 2007 (EDT)

6/14/07

I obtained the cultures that I had grown overnight and miniprepped the cultures from colonies hemA1-hemA3 and hemC1-hemC3. I then took 5ul from each miniprep and digested with Bg1II and Xho1 to see if this digestion would separate my plasmid into parent vector and insert. When I did this and tested on a gel, I saw my gel had been smeared strangely. It looked as if my A1 and A2 were squished together, while the others were ok. Just in case, I chose A3 and C1 to send for sequencing. This time, I sent in two samples of each miniprep and asked quintara to sequence with both ca998 and G01001 so I can analyze the entire sequence of my gene. Afterwards, I analyzed the sequencing results of B4 and D4 with G01001. After some struggling, I confirmed that the last 600-700bp matched up using the new sequencing results. Even though the first 100 or so were a little off with these sequencing results because the primer can only go for so long, I knew my B4 and D4 were fine because the sequencing results of the day before showed the beginning 600-650 bp are good. Kdoan 18:20, 28 June 2007 (EDT)

6/15/07

I began with looking at my sequencing results for A3 and C1. Both had many mutations when compared to the predicted PCR products. I discarded these results assuming they were faulty and sent in A2 and C2 to see if these clones were matches. Again, I sent in two samples of each and included ca998 and G01001 because these genes are too large for just the reverse. I then began the process of adding the RBS to hemB4 and hemD4. I made construction files for the composite part RBS + hemX and obtained the RBS. I digested the RBS with BamH1 and XhoI and the heme genes with Bg1II and XhoI. I ran my digestions on a gel and excised the bands. However, at this point, I discovered I was given the wrong RBS. I was given a pooled sample of RBS, while what I needed is pBca1101-I716051-kan cassette. So I drew up new construction files for the correct RBS composite part. I was able to keep the gel bands for hemB and hemD, but I had to re-digest the new RBS I received with BamHI and XhoI. After I waited for the digest and excised the bands for the rbs, I purified the two RBS, hemB, and hemD digestion products. Kdoan 18:23, 28 June 2007 (EDT)

6/17/05

Yes, I came in on a Sunday. No one else was here except for John for an hour. During my stay, I began the cloning process for hemA from CFT073. All my previous attempts had failed because I was adding 1uL of buffer instead of 5uL. I obtained more CFT073 from Chris. This hemA from CFT073 also has an internal restriction sites so my first PCR reactions were to eliminate this Bg1II site. One PCR reaction included oligos kd013/kd016 and another included kd015/kd014. Afterwards, I ran my PCR products on a gel, excised the bands, and purified the gel bands. I then ran a second PCR to combine these two fragments to get the entire hemA gene from CFT073 and left the PCR tube in the machine overnight. I then resumed with the making of the composite part of RBS+hemB and RBS+hemD. I had digested these, ran them on gels, excised the bands, and cleaned up the gel bands. I then ligated RBS+hemB and RBS+hemD and transformed in DH110B competent cells. Because this RBS has a kan cassette and not a carb, I had to add 100uL of LB media and put the transformation reactions in the incushaker for one hour for the cells to start production of kanR so they wouldn't die immediately on the LB+kan plates. I then plated each transformation reaction on two plates and left them overnight to grow. Kdoan 18:23, 28 June 2007 (EDT)

6/18/07

My two plates of RBS (pBca1101-I716051-kan cassette) + hemB and RBS + hemD had colonies so I chose three from each (hemB+rbs1-3 and hemD+rbs1-3 and grew them in LB+carb media. It would've been better if I grew these colonies in LB+kan, but the RBS I used also has an AmpR gene so my cells will still live. I worked around a flame so there should be little chance of contamination. Afterwards, I obtained the PCR reaction I left overnight of hemA from CFT073, ran an analytical gel and saw a product. I then cleaned the PCR product, digested with Bg1II/XhoI/DpnI, ligated with pBca9145-1144#5 vector, and transformed into DH110B competent cells. I then plated and left it in the 37C incubator overnight. I then proceeded with looking at the sequencing results I received over the weekend. Both A2 and C2, like A3 and C1, had mutations. However, upon close inspection, I noticed that the mutations of C1 and C2 were identical. Two different GC-->CG mutations in the same position. Otherwise, these clones were fine. I looked up the sequence for hemC I found on colibase and compared them to pubmed. These differences matched the differences I noticed with my clones. My clones matched perfectly with the ones in pubmed. So both my C2 and C1's worked. I then turned to A2 and A3. A3 had many mutations. A2 also had a GC-->CG mutation which matched with two of the many mutations I saw in A3. I couldn't find another hemA R.capsulatus sequence that could verify that I was working with a different template from the one displayed in pubmed. I consulted Chris and he said with 90% assurance that the mutations were OK to work with since both clones had the same mutations. Also, I asked Richard from Quintara to redo my A2 and C2 with G01001 because they sequenced with ca998 and I need to analyze the second half of my gene. Kdoan 18:21, 18 June 2007 (EDT)

6/19/07

After growing my hemB+rbs1-3 and hemD+rbs1-3 in culture, I miniprepped all three and began to run them on test gels to make sure that when I cut with BglII and XhoI I would see one band corresponding to insert and one band corresponding to parent vector. However, Chris then informed me that I had done them incorrectly. Instead of cutting out the RBS and putting it in front of the heme gene, I cut out the heme gene and put it behind the RBS. So even though the two were in the correct order, the parent vector pBca9145 would not be maintained. So I decided to just wait until I had all five of my heme gene basic parts ready (hemA from CFT073, hem A from R.capsulatus, hemB, hemC, and hemD) and add the RBSs all at once. I then also learned that I had to add two different kinds of RBS to each heme gene. One RBS library to each and one single RBS to each. I chose pBca1101-1106B as the single RBS to add to each heme gene. As for the RBS library, I need to alternate between pBca1101-I716051, which has a kan cassette, and pBca1101-I716052, which has a cam cassette. So hemA from CFT073, hemA from R.capsulatus, and hemC will get the RBS library with the kan cassette and hemB and hemD will get the RBS library with cam casette. I planned out exactly how I was going to do this. Afterwards, I chose three colonies from the hemA from CFT073 plate and grew these in culture. Then I analyzed my sequencing results. A2 and C2 with G01001 and ca998 looked great and these are the colonies that I will add RBS's to. Kdoan 18:22, 28 June 2007 (EDT)

6/20/07

I decided to postpone the intense RBS adding until I had all five of genes sequenced and ready to go. So I miniprepped the three colonies from hemA from CFT073, digested 5uL from hemAC1, hemAC2, and hemAC3 with BglII and XhoI, and ran these samples on gel. They all looked good so I chose to send hemAC1 and hemAC2 for forward sequencing with ca998 and reverse with G01001. Austin told me we had few pBca1101-I716052 and pBca1101-I716o51 left, so he handed me one culture of each and I miniprepped 50uL more of each. Since I had a free day afterwards I made more gels and helped set up some shelfs for the new fridge. I then transferred some bottles and plates from the old frigde to the new one. I checked over my plans and made sure I was ready to go for the next day. Kdoan 18:21, 28 June 2007 (EDT)

6/21/07

Long day. I had 13 digestions going on. Five for the heme genes being cut with EcoR1/BglII to add to the RBS libraries, five for the heme genes being cut with BglII/XhoI to add to the single RBS, and three for each kind of RBS (one for I716051, one for I716052, one for 1106B). I digested for an hour. Ran all of them on gels. Extracted the bands and gel purified. Afterwards, I ligated with their appropriate pairs and transformed into DH110B competent cells. The five being added to the single RBS 1106B I was able to plate right away on LB-Amp plates, but the other ones I had to add 100uL of LB media and grow in the incushaker for about 30 minutes. Afterwards, I plated hemA from R.capsulatus, hemA from CFT073, and hemC on LB/Amp/kan plates because these were ligated to the RBS I716051 with kan resistance while hemB and hemD were grown on LB/Amp/Cam plates because these were ligated to the RBSI716052 with cam resistance. I grew these in the 37C incubator overnight. Kdoan 18:22, 28 June 2007 (EDT)

6/22/07

Lab got flooded today! Disaster! People moved our plates from the 37C incubator to the fridge. I came in for a bit to check out the situation. I also looked at my plates and all had colonies. Kdoan 18:21, 28 June 2007 (EDT)

6/25/07

I chose two colonies from each plate. So I had twenty tubes. The 1106B RBS's I was able to grow in just LB-Amp culture, but the I716051 RBS's I grew in LB-Amp-Kan and the I716052 RBS's I grew in LB-Amp-Cam. Kdoan 18:21, 28 June 2007 (EDT)

6/26/07

I discovered that for the I716051 and I716052 RBS libraries I'm not supposed to just pick single colonies and grow these in culture. I'm supposed to add 2mL of LB media to each plate, spread until the mixture is homogenous, extract 1uL of mixture and grow this 1uL in the appropriate LB-antibiotic media. So I threw away the ten cultures I grew up overnight that corresponded to single colonies I picked from the gene+RBS library plates. Then I grew up the RBS library colonies the proper way in five culture tubes with the appropriate LB-antibiotic media. I retrieved my ten culture tubes and began to miniprep. I noticed that almost all of them had a pinkish tint, but didn't look into it. When I transferred some liquid to centrifuge tubes and pelleted the cells, I noticed the cells were pink. I knew that this was from RFP. When I looked at my construction files, I saw that if my digestions had worked properly, the RFP should've been cut out. I consulted Amin, and he said that my digestions were probably inadequate and next time I should wait for at least two hours. Chris checked my construction files and everything should work ok. I noticed there wasn't enough 1106B RBS so I decided to use 1106A so I changed my construction files. I prepared for the next day and made more agarose gels. Kdoan 18:20, 28 June 2007 (EDT)

6/27/07

This time I ran my digestions for two hours. I ran them all on gels. However, rbs I716051, 1106A, hemC digested with EcoR1/BamH1 and hemD cut with Bg1II/XhoI were still inadequately cut. Chris suggested I mix more thoroughly. I extracted the other bands and melted them in ADB buffer and refrigerated. I set up the digestions that didn't work as well and during this time, we had our meeting. Afterwards, I ran the re-digestions on a gel, gel extracted, and purified all thirteen digestions products and eluted each with 10uL of water. Now they're ready for ligations tomorrow. Kdoan 18:20, 28 June 2007 (EDT)

6/28/07

I ligated the appropriate RBS with its corresponding heme gene. I then transformed into DH110B competent cells. The ones ligated to 1106A RBS I plated immediately on LB-Amp plates, but the RBS libraries I grew in 100uL of media for one hour then plated them onto their appropriate plates. I made more gels and updated the registry with my parts. I also noticed that my hemB4 and hemD4 were running low and just in case my RBS additions failed again, I would need more of these minipreps. So I picked more of these colonies and grew them in culture once more so tomorrow I could miniprep. I also helped out Sam by gel purifying two of her samples and David with autoclaving tips. Kdoan 18:20, 28 June 2007 (EDT)

6/29/07

I retrieved my ten plates. I need to wait longer before the colonies with parent vector turn pinkish-red from RFP. I miniprepped the hemB4 and hemD4. I picked colonies from Vai's plates and grew them in 12 tubes with LB-Amp culture. I also made more LB-Carb media because the stock was running low. I then chose colonies to grow in culture from the ten plates. Well, for the five RBS libraries, I added 2mL of LB media, spread this around, then grew 1uL of the mixture in the appropriate LB+antibiotic media. For the other five plates with the single RBS 1106A, I chose two colonies from each. I looked at the plates under the UV lamp to make sure the colonies I chose did not glow pinkish from the RFP gene. I then grew these clones in LB-Amp media. Kdoan 15:15, 29 June 2007 (EDT)

6/30/07

I came in on a Saturday! I retrieved my fifteen culture tubes from the incushaker. Upon initial inspection, I noticed that 1106A+hemC1 was the only one that looked pink, most likely these DH110B cells had taken up uncut 1106A parent vector, which also has the RFP gene. The 1106A+hemA1 and 1106A+hemA2 looked quite brownish and the 1106A+hemAC1 and 1106A+hemAC2 was a lighter brown. These browns were distinctly different from the pink from the RFP. I pelleted the cells and as I suspected, the 1106A+hemC1 was pink from the RFP and it fluoresced when viewed under UV light. The 1106A+hemA1/2 and 1106A+hemAC1/2 were brownish (1106A+hemA1/2 was darker brown)and Chris and I believe that this is from the production of heme. Heme has a brownish/red phenotype so hopefully this brown pellet is from the accumulation of heme. I then completed the 15 minipreps. Afterwards, I moved Vai's culture tube and two transformation plates from the incubator room to the fridge. Kdoan 14:55, 2 July 2007 (EDT)

7/2/07

I do not need to send the RBS libraries in for sequencing, but the single RBS 1106A I will send for sequencing. Even though the nine 1106A+gene clones did not become pink and so they do not contain uncut RBS parent vector, they can still contain uncut pBca9145-gene parent vector. So I ran a test digest where I cut these minipreps with Bg1II/XhoI and ran the nine minipreps on a gel. However, I noticed this lane of gels I made the wells cut all the way through the bottom. So my gels were too thin for how low the combs were. I checked to make sure the other gel lanes didn't have this problem. Some of my samples ran out but I hoped there was enough DNA to detect. The ones that didn't run out had the correct size bands but there were three lanes (hemA2, hemAC1, hemAC2), which had nothing appear and coincidentally, these were the same ones where my DNA samples had run out. I decided to send one from each gene+1106A RBS sample anyway for sequencing. I sent hemA1, hemAC2, hemB2, hemC2, and hemD2 for forward sequencing with ca998. I then made construction files to link together hemA+hemB, hemAC+hemB, and hemC+hemD for both the single and library RBS. Kdoan 14:55, 2 July 2007 (EDT)

7/3/07

I looked at my sequencing results. RBS+ gene for HemA1, hemAC2 hemB2, and hemD2 were good except they had two identical mutations. These came before the RBS+gene and after any restriction sites so I assumed these were ok. HemC2 Richard said did not work, but I proceeded anyway. I followed my construction files to set up the ten digests. Five from the libraries and five from the single RBSs. After I put the digests in the 37C, I realized that for the libraries, the way in which I digested would only keep the kanR gene from hemA, hemAC, and hemC, but cut out the CamR gene from hemB and hemD. Chris had told me to alternate antibiotic resistance genes and I figure that would be for a reason. So I emailed him to ask about this and decided to wait on the libraries until I know exactly what I am supposed to do. After two hours, I ran my digests on a gel, but did not get bands for hemC2 cut with BamHI and XhoI. So I redid this digest and cut out the other ones and gel purified. After another two hours, I ran hemC2 again on a gel but still did not get bands. Maybe I did my miniprep wrong and that is why quintara had difficulty getting sequencing results. I still had the digested hemC with Bg1II/XhoI and RBS 1106A with BamHI/XhoI so I decided to re-ligate and transform this since my 1106A+hemC plate had only 7 colonies and only one was white. So I did this transformation along with the hemA+hemB and hemAC+hemB. Kdoan 14:55, 2 July 2007 (EDT)

7/4/07

I looked at my plates. For some reason, hemA+hemB did not have any colonies. I still have the digested/purified hemA as well as the digested/purified hemB, so I will just re-ligate and transform tomorrow. I chose two colonies from the 1106A+hemC and hemAC+hemB plates and grew these in LB+Amp media. Kdoan 17:01, 5 July 2007 (EDT)

7/5/07

The two colonies I chose from 1106A+hemC turned pink from the RFP. I looked at the original plate and all the colonies were pink. I threw this plate away. I can't use the digested/purified hemC because this contains too much undigested parent vector. I'll have to re-digest hemC2 tomorrow and ligate this to 1106A RBS. I digested the RBS libraries+gene to make composite parts (hemA+hemB, hemAC+hemB, and hemC+hemD). When hemA is cut with EcoR1/BamHI/ScaI, there are 1.5kb and 1.27kb bands which will be hard to resolve so I'll need to run the gel slow and add EtBr to the buffer. After 2-hr digestions, I ran the samples on a gel and gel extracted, but I didn't get bands for hemA. I gel extracted and purified the others and ligated them. I then transformed into Dh110B cells and then added 100uL of LB media to each transformation and incubated for 30 minutes because I'll be plating on LB+Amp+Kan plates. I then plated the hemAC+hemB and hemC+hemD transformations. During the two hour digest, I also miniprepped the two colonies I picked from hemAC+hemB (single 1106A RBS), made more gels, and re-ligated and retransformed the single RBS hemA+hemB since I didn't get any colonies the first time. The two minipreps I digested 5uL with Bg1II/XhoI to see if I would get the correct bands assuming my cassette is kanR-RBS-hemAC-RBS-hemB. I then ran these on a gel, but my bands were very faint so I ended up sending both for forward and reverse sequencing anyway. Kdoan 14:55, 2 July 2007 (EDT)

7/6/07

Both the minipreps for single RBS hemAC+hemB for forward and reverse sequencing were good. I set up digests for single RBS hemA and library RBS hemA to string them together with the single RBS hemB and library RBS hemB that I already have purified/digested. Unfortunately, the plates I did for library RBS hemAC+hemB and library RBS hemC+hemD did not yield enough colonies for a library. They did get maybe 20 colonies for each plate. Because I still have the purified/digested products for library RBS hemX, I just need to re-ligate and re-transform. I did plasmid transformations for hemA2, hemC2, Bca1101-1106A, and Bca1101-1106B because these minipreps are running low. I also made more gels. After single RBS hemA and library RBS hemA digested for two hours, I ran these samples on a gel and extracted the appropriate bands. I cleaned up the digest, ligated, and transformed into DH110B cells. I plated the single RBS hemA+hemB right away but the other ones I incubated in 100uL of LB and plated on LB+Amp+Kan plates. Kdoan 14:55, 2 July 2007 (EDT)

7/7/07

I came in really quickly to grow things in culture. For the ShemA+ShemB plate, there was only one colony. Chances are it won't be right, but I decided to grow this one colony in culture anyway. The LhemAC+LhemB plate had only about 15 colonies so I'll have to redo this one. The LhemA+LhemB had 30 colonies so I added 2mL of LB, spread the media around, then grew 1uL in LB+Amp+Kan media. The 1106A, 1106B, hemA2, and hemC2 plates had plenty of colonies. The 1106A/B plates had pink colonies as expected. So I just chose one colony from each and grew these in LB+Amp media. Kdoan 17:25, 9 July 2007 (EDT)

7/8/07

I retrieved my cultures from the incushaker and miniprepped all seven tubes. The minipreps for 1106A/B I put back into PB#3. The hemA2, hemC2, ShemA+ShemB1 and LhemA+LhemB minipreps I put in my miniprep box and I sent ShemA+ShemB1 for forward and reverse sequencing. Kdoan 17:25, 9 July 2007 (EDT)

7/9/07

I digested hemC2 in order to ligate it to single RBS 1106A. I also digested 51+hemAC in order to make the composite part (RBS library) hemAC+hemB. I made gels during this time. After two hours, I ran the two samples on a gel, extracted the appropriate sized bands, and purified. I then ligated hemC2 to single RBS 1106A and 51+hemAC to 52+hemB. Single RBS 1106A and 52+hemB were previously digested and purified. I transformed into DH110B cells and plated hemC2+1106A on LB+Amp plates. LhemAC+LhemB I had to incubate for one hour in 100uL of LB media because I plated this transformation on LB+Amp+Kan plates. Kdoan 17:43, 9 July 2007 (EDT)

7/10/07

The plates I transformed were no good. The LhemAC+LhemB had only about 10 colonies, which is definitely not enough. The 1106A+hemC2 plate had four colonies that were already turning pink. I checked my sequencing result for ShemA+ShemB1, and the sequence contained only RBS+hemA and not RBS+hemB. Chris suggested I use 4uL of each ligation product instead of one and transform into TG1 cells instead of DH110B cells. Since I still had the purified/digested products required for each ligation, I just religated and transformed. The 1106A+hemC2 and ShemA+ShemB I plated on LB/Amp plates, but the LhemAC+LhemB I rescued and plated on LB+Amp+Kan plates. I digested LhemA+LhemB and LhemC+LhemD to make the composite part hemA+hemB+hemC+hemD. I also digested these composite part libraries to check to see if I would get the correct band sizes. After a two hour digestion, I ran an analytical gel and got the right band sizes for LhemA+LhemB and LhemC+LhemD. However, my other gel containing the digestions to make my super composite part failed. I got more bands than I expected for each digestion, showing that I had incomplete digests. I will try the whole procedure again tomorrow. Kdoan 17:56, 10 July 2007 (EDT)

7/11/07

The ShemA+ShemB plate had four colonies. The LhemAC+LhemB had a lawn. 1106A+hemC2 had many colonies, most of which were pink, but about ten were white. I added 2mL of LB to LhemAC+LhemB, spread, and grew 1uL in LB+Amp+Kan culture. I chose all four ShemA+ShemB and four of the ten white colonies from 1106A+hemC2 and grew these in LB+Amp. I digested mini LhemA+LhemB and LhemC+LhemD to string these composite parts together. I made gels during the two hour digestion. I ran the digestions on a gel and saw the same results as yesterday. In addition to the bands I expected for both samples, for LhemA+LhemB I got a smear of DNA below the smallest band. Amin says this must be uncut vector DNA. Luckily, I needed the top band, which seemed to correspond to the 3.3kb band I wanted. The LhemC+LhemD got the two bands I expected as well as two other ones. I cut the largest band, which also seemed to correspond to the 3.8kb band I wanted. I cleaned these up, ligated them (4uL of each ligation product again), and transformed into TG1 cells. I rescued for one hour then plated on a LB+Amp+Kan plate.

7/12/07

I came in at 12:30AM and grew the LhemA+B+C+D in LB/Amp/Kan culture. When I came in later, I miniprepped the samples I grew in culture yesterday. The pellets of LhemAC+LhemB were pretty brown and so were ShemA+ShemB1 and 4. I decided to send 1106A+hemC3/4 for sequencing because these were slightly brown compared to the other two. I set up digests to combine LhemAC+LhemB and LhemC+LhemD. I also digested the ShemA+ShemB1-4 and LhemAC+LhemB with BglII/XhoI to see if I got the correct sized bands. I ran a gel and extracted the bands. Again, I saw four bands (???) for LhemC+LhemD and extracted the 3.8kb band. For the test digests, ShemA+ShemB4 looked the best and LhemAC+LhemB had the band sizes I expected. I gel purified LhemAC+LhemB and LhemC+LhemD, ligated them with 4uL of each, transformed into TG1 cells, rescued for one hour, then plated on an LB/Amp/Kan plate. During this time, I sent kd031-kd034 for sequencing and miniprepped LhemA+B+C+D and ran a test digest w/ BglII/XhoI. I expected a 4kb and 3kb band, but got 3 and 2kb bands instead. I suspect this might be because I cut the wrong band for the LhemC+LhemD digest, which mysteriously gave me four bands three times. Maybe I extracted uncut LhemC+LhemD plasmid. I'll try again. I suspected something was wrong because the pellet of LhemA+B+C+D was not brown. Kdoan 18:30, 12 July 2007 (EDT)

7/13/07

I grew the LhemAC+B+C+D in LB/Amp/Kan culture even though I think it's wrong since the LhemC+LhemD got four bands and I extracted the top band again. The same thing as the LhemA+B+C+D will probably happen so just in case, I redid both LhemA+B+C+D and LhemAC+B+C+D. This time, LhemC+LhemD had two bands, and the correct ones! So I gel extracted and purified. I ligated, transformed into TG1 cells, rescued for one hour, then plated on a LB/Amp/Kan plate. I also made gels and helped David with autoclaving tips. Quintara did not give me results for 1106A+hemC3/C4 so I digested both. Only 1106A+hemC4 gave me the correct band sizes so I ligated this product to ShemD, which I had purified/digested previously and transformed into TG1 cells again and plated immediately on a LB/Amp plate. Kdoan 13:14, 16 July 2007 (EDT)

7/14/07

I came in briefly to pick four colonies from the ShemC+ShemD plate and grow these in LB/Amp culture. I also grew 1uL of both LhemAC+B+C+D2 and LhemA+B+C+D in LB/Amp/Kan culture. I moved the culture of LhemAC+B+C+D1 that I grew overnight on Fri to the fridge so I can miniprep all my cultures at once on Sun. Kdoan 13:17, 16 July 2007 (EDT)

7/15/07

I miniprepped all eight culture tubes. I noticed that some of my libraries did not have brown pellets at all and the ones that did were a light brown color. I'll run a test digest with Bg1II/XhoI on Mon and I'm prepared to have to do the process again. Kdoan 13:20, 16 July 2007 (EDT)

7/16/07

I ran test digests of LhemA+B+C+D and LhemAC+B+C+D1/2. These should give me fragments of 4 and 3kb. In conjunction with these test digests, I ran digests of LhemA+B, LhemAC+B, and LhemC+D so I can begin stringing the genes together again just in case the test digests show me bad results. I made more gels and after two hours, I ran my samples on a gel. My test digests showed that the 4 and 3kb bands were there, but in addition to these correct bands, the LhemA+B+C+D had an extra band below (possibly uncut vector) and the two LhemAC+B+C+D samples also had two extra bands. I figured these extra bands are background since I am working with a library and at least the bands I want are there. I decided to ligate and transform the digests I ran and I'll just have multiple LhemA+B+C+D and LhemAC+B+C+D copies to assay. I extracted the bands from the new digests, purified, ligated, transformed into TG1 cells, rescued for one hour, and plated on LB/Amp/Kan plates. Kdoan 16:44, 16 July 2007 (EDT)

7/17/07

Now that I have my LhemA+B+C+D and LhemAC+B+C+D parts, I now have to add David's T7 promoter in front. I digested LhemA+B+C+D and LhemAC+B+C+D1/2 with EcoR1/Bg1II for two hours and ran these on a gel. The LhemA+B+C+D got 4 and 1kb bands even though I was expected 6 and 1kb bands. The LhemAC+B+C+D1/2 got the bands I expected plus two other bands below the 6kb band. So I gel extracted LhemAC+B+C+D1/2 and gel purified, but I also repeated the same digests for all three samples for two hours. When I ran a gel, I got the same result. So my LhemA+B+C+D is no good and I gel extracted the 6kb band again from the LhemAC+B+C+D1/2. I purified these and ligated my products from the first digest with the T7 wildtype promoter. I transformed into TG1 cells and plated onto LB/Amp plates. Kdoan 16:35, 17 July 2007 (EDT)

7/18/07

I came in to pick three colonies from each of my LhemAC+B+C+D1/2 + T7 plates and grow in culture so that I can test if the T7 promoter as well as all four of my heme genes are there by PCR. I then scraped the plate and extracted 70uL from each plate to miniprep and perform plasmid transformation into BLR cells. Along with these minipreps, I also miniprepped LhemAC+B+C+D3 and LhemA+B+C+D2. I also cleaned up a digest for Vai and then we all went to the baseball game. Kdoan 15:50, 19 July 2007 (EDT)

7/19/07

Chris suggested that because my single RBS genes have been consistently more brown than the library RBS genes I should really concentrate more on the singles instead. In conjunction with this, I will backtrack to 51 library RBS+hemA, pick the clone that yields the brownest product and begin connecting hemB, hemC, and hemD from there. On the plus side, Chris told me that the single RBS 1106A already has a promoter on it. This explains why I've been getting brown products with the single RBS from the beginning. With regard to the single RBS's, I have hemA+hemB and hemAC+hemB, but I haven't been able to make the composite part 1106A+hemC. I've had multiple failed attempts so I digested 1106A and hemC, gel extracted, purified, ligated, transformed into DH10B cells, and plated on LB/Amp. I made gels during the two hour digest and performed plasmid transformation for 51+hemA and 51+hemAC into TG1 cells and plated on LB/Amp plates. Kdoan 16:25, 19 July 2007 (EDT)

7/20/07

I went home to SJ for the weekend. I asked Austin to put my RBS 51 + hemA and RBS 51 + hemAC in the fridge and to select four non-pink colonies from the 1106A+hemC plate and grow them in LB/Amp culture. Kdoan 15:41, 23 July 2007 (EDT)

7/21/07

I asked Austin to take the four culture tubes (1106A+hemC1-4) from the incushaker and put these in the fridge. Kdoan 15:43, 23 July 2007 (EDT)

7/22/07

I came in today to make up for my absence on Fri. I looked at my four 1106A+hemC culture tubes and noticed two were pink from RFP. I discarded these and kept the other two to miniprep on Mon. I decided to ligate and transform the remaining digested/purified single RBS 1106A and hemC2 so I could sent more for sequencing in case 1106A+hemC1 and 1106A+hemC4 do not work. There were 6uL of each digested/purified product left and I transformed into TG1 cells and plated on LB/Amp plates. As for my plates RBS 51+hemA and RBS 51+hemAC, I decided to grow 96 clones in a 96-well block so I can select for single RBSs in the library that turn the cells most brown. I had to autoclave the two 96-well blocks. I put 1mL of LB/Amp media in each well and picked individual clones to grow in each well. Then I covered them with sticky gauze and put them in the special incushaker upstairs. Kdoan 15:48, 23 July 2007 (EDT)

7/23/07

I obtained the 96-well blocks from the incushaker and pelleted the cells. When I looked at the pellets, I noticed the clones from RBS 51+hemAC were browner than the ones from RBS 51+hemA. Some were lighter than others, but many were equally browner. So I just chose five of the browner ones from each plate, pipetted up and down to resuspend the cells, and extracted 1uL to grow in 5mL of LB/Carb/Kan media. I also grew one clone from an old hemC2 plate in 5mL of LB/Amp to act as a negative control so that tomorrow I can compare the brown w/ the non-brown and make sure there is a noticeable difference. I picked three clones (1106A+hemC2/3/5) from the new 1106A+hemC plate and grew these in LB/Amp culture and miniprepped the 1106A+hemC1/4 from before. I ran a test digest of 1106A+hemC1/4 with Bgl11/XhoI in parallel with a digest to begin combining these with 1106A+hemD, which I also set up a digest for. Unfortunately, both 1106A+hemC1/4 had the wrong sized bands so I did not combine 1106A+hemC with hemD. So I redigested hemC1 clone and 1106A, ran a gel, got good bands, gel extracted, ligated, transformed into TG1 cells, then plated on an LB/Amp plate. Kdoan 16:00, 24 July 2007 (EDT)

7/24/07

I picked four colonies from the 1106A+hemC plates and grew these in LB/Amp culture. I compared the pellets from hemC2 with the five singles from 51+hemA and 51+hemAC. The 51+hemA and 51+hemAC were considerably darker. I picked one of each of the five singles and grew another 1uL in LB/Amp/Kan and I will compare these to 1106A+hemA/hemAC. Meanwhile, I miniprepped one single from 51+hemA and one from 51+hemAC. I digested each with EcoR1/Bg1II, ran gels, gel extracted, purified, ligated these with T7 promoter that has already been digested with EcoR1/BamH1, transformed into TG1 cells, rescued for one hour, then plated on LB/Amp/Kan plates. At the end of the day, I miniprepped the 1106A+hemC1-4 after only 5 hrs in the incushaker because I needed to send these in for sequencing asap. Since the pellet was smaller I eluted in 40uL of water for the miniprep. Kdoan 15:51, 26 July 2007 (EDT)

7/25/07

I compared the pellets of 1106A+hemA/hemAC and 51+hemA and 51+hemAC. The 51+hemAC was darker than the 1106A+hemAC, while the 1106A+hemA is darker than the 51+hemA. It's not a fair comparison though since the 51+hemA/AC do not have promoters on them, while the 1106A has the Pcon promoter attached. I decided to go on with both experiments in conjunction. I added 2mL of LB to the T7+51hemA and T7+51hemAC plates, scraped, and extracted 70uL of mixture to miniprep. I miniprepped and did a plasmid transformation into 50uL of BLR cells and plated on LB/Amp/Kan plates. I checked my sequencing results. 1106A+hemC1/2 did not even have a ca998 binding site said Richard, 1106A+hemC3 had RBS then random sequence again, and 1106A+hemC4 was just hemC parent vector. I finally decided to try DBBS. I set up the two PCRs for DBBS. The first time, the PCR for 1106A worked, but hemC got no band. I set up a second PCR for hemC and left it overnight. I did the transformation of T7+51hemA/AC into BLR again just in case. Kdoan 16:16, 26 July 2007 (EDT)

7/26/07

For the second PCR reaction for hemC, I got two bands and none of them corresponded to the right size band. I set up yet another one for hemC, but at the same time, I transformed 1106A and hemC into lefty and righty strands so I can try John's method. I autoclaved two 96-well blocks, let them cool for some time, added 1mL of LB/Amp/Kan media to each well, and picked colonies from my T7+hemA and T7+hemAC in BLR cells to grow in each well. I brought the blocks upstairs to shake and left them overnight. I made more gels. In the end, I checked my third hemC PCR by an analytical gel and this one yielded no bands again. So I will move on to John's method. Kdoan 12:57, 30 July 2007 (EDT)

7/27/07

I realized I made the mistake of plating T7+hemA and T7+hemAC in both TG1 and BLR on LB/Amp/Kan plates. When I grew colonies in the 96-well blocks, I also grew on LB/Amp/Kan plates. So the clones I had been working with were uncut 51+hemA and 51+hemAC vectors because when cut with EcoR1 and Bg1II, the KanR gene is eliminated. I discarded all these plates and cleaned out the 96-well blocks. I began digesting 1106A hemA+hemB and 1106A hemAC+hemB and 1106A hemD so I can make this composite part for now and begin assaying by next week. I will complete the part 1106A+hemC and make the full composite part asap. I couldn't find the miniprep for 1106A+hemD so I miniprepped from an old culture hoping that it's still good to work with. After two hours, the digestion 1106AhemA+hemB yielded three bands while the 1106AhemAC+hemB yielded no bands. I cut out the band that seemed correct from 1106AhemA+hemB and surprisingly, the 1106A+hemD yielded the two correct bands. I cleaned up, ligated, and transformed into TG1 cells and plated on LB/Amp. At the same time, I ligated pre-digested/purified products of 51+hemA and 51+hemAC with the T7 promoter and transformed into TG1 and plated on LB/Amp plates this time. I picked one colony from each of the lefty 1106A and righty hemC plates and grew these clones in LB/Amp cultures. Kdoan 13:10, 30 July 2007 (EDT)

7/28/07

I added 2mL of LB media to the T7+hemA and T7+hemAC, scraped, and extracted 700uL to miniprep. I miniprepped these two samples along with the lefty 1106A and righty hemC. During the ten minute centrifuge with P1, P2, and N3, I picked eight colonies from the ABD plate and grew them in LB/Amp cultures. After the minipreps, I transformed 2uL of T7+hemA and T7+hemAC into BLR cells and plated on LB/Amp plates. Kdoan 12:57, 30 July 2007 (EDT)

7/29/07

I came in very briefly to parafilm my T7+hemA and T7+hemAC in BLR plates and move them to the fridge. I also transformed my ABD1-8 cultures tubes to the fridge. Kdoan 12:57, 30 July 2007 (EDT)

7/30/07

I set up digestions for lefty 1106A and righty hemC. During this two-hour digestion, I miniprepped ABD1-8. All of the pellets were the darkest brown I've seen yet. One was significantly lighter brown (ABD2) so I discarded this one immediately. I autoclaved two 96-well blocks and added 1mL of LB/Amp to each well. I picked colonies from T7+hemA in BLR and T7+hemAC in BLR. The T7+hemA did not have enough colonies to fill the 96-well block, so I left about three rows empty. I covered the blocks and placed them in the incushaker upstairs and then made gels. I then ran my digests on a gel, gel extracted, cleaned up, ligated, and then heat-killed the ligase for 20min in the PCR machine set at 65C block. I then added BamHI and BglII to eliminate any uncut vector and ran this digest for one hour. Then I transformed into TG1 cells and plated on LB/Amp plates. I also set up test digests with BglII/XhoI on ABD1 and ABD3-5. After one hour, I ran these samples on a gel. However, my bands corresponded to uncut vector 1106A hemA+hemB. I decided to try John's lefty/righty method with this composite part as well so I transformed 1106A hemA+ hemB as well as 1106A hemAC+hemB into the lefty strain and 1106A hemD into the righty strain. Kdoan 17:45, 30 July 2007 (EDT)

7/31/07

I came in to pick one colony from each of the lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, and righty 1106A hemD plates and grow these in LB/Amp cultures tubes. My 1106A+ hemC had only three colonies! So I picked all three and grew in LB/Amp cultures as well. Austin suggested I look up the toxicity of hemC since I'm having SOO much trouble, but hemC doesn't appear to have any toxicity. I retrieved my T7+hemA in BLR and T7+hemAC in BLR blocks and spun them down. T7+hemA had around five clones that were very dark brown. T7+hemAC had about a third of the clones that were browner than the rest but much less brown than the T7+hemA. I picked two of the darker ones from each block and grew 2uL of each in LB/Amp cultures. I also made lots of construction files and looked up an old heme article to find out about the LCMS they used to quantify the amount of heme being expressed by their cells. I emailed the article to Mario so he can see if it's doable. Kdoan 18:03, 31 July 2007 (EDT)

8/1/07

I came in the night to miniprep lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, righty 1106A hemD, 1106A+hemC1-3, T7+hemA1-2 and T7+hemAC1-2. I came in the following morning and sent 1106A+hemC1-3 for sequencing. Then I digested righty 1106A hemD, lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, T7+hemA2, T7+hemAC2, and 52+hemB so I can make composite parts. I made gels and after a two-hr digestion, I ran a gel. The 1106A hemA+hemB, 1106A hemAC+hemB, and 52+hemB worked so I gel extracted these, added ADB, and melted the gels. Righty 1106A hemD yielded no bands, while T7+hemA2 and T7+hemAC2 yielded too many. I set up second digestions, but used T7+hemA1 and T7+hemAC1 instead. After two hrs, righty hemD still yielded no bands, while T7+hemA1 got the correct bands and T7+hemAC1 yielded too many bands again. I figured there was no DNA in the miniprep of righty hemD I was working with and grew another clone of righty hemD in LB/Amp culture to miniprep again tomorrow. Kdoan 16:51, 3 August 2007 (EDT)

8/2/07

I miniprepped righty hemD and ran digestions of righty hemD, T7+hemAC1, and T7+hemAC2, hoping at least one would work. After two hours, righty hemD worked, while the T7+hemAC1 and T7+hemAC2 yielded only one band. I gel extracted righty hemD and cleaned up the digestion along with the digestions that worked from yesterday. I ligated the respective products together. I transformed T7+hemA + hemB immediately on LB/Amp plates. The lefty and righty ones I heat-killed the ligase at 65C for 20 min, digested with BamHI and Bg1II to get rid of parent vector, and transformed into TG1 on LB/Amp plates. Kdoan 16:51, 3 August 2007 (EDT)

8/3/07

I chose four colonies from each 1106A hemABD and 1106A hemACBD and grew these in LB/Amp cultures. I then added 2mL of LB media to the T7+hemA + hemB plate and scraped. I then extracted about 1mL and miniprepped. I then performed a plasmid transformation into 90uL of BLR cells and plated on an LB/Amp plate. I helped David autoclave. Kdoan 16:59, 3 August 2007 (EDT)

8/4/07

My T7+hemA+hemB plate came out very weird. There were lots of colonies but tons of satellite colonies as well. So I took the miniprep of T7+hemA+hemB in TG1 and did another plasmid transformation into BLR cell on LB/Amp plates. Just in case the LB media I used was contaminated, I took the digested/purified products of T7+hemA and hemB and religated and transformed into DH10B cells. I then plated on an LB/Amp plate. I also miniprepped the four 1106A hemABD and four 1106A hemACBD. I only kept three of the 1106A ABD and two of the 1106A ACBD. I can tell from pellet color that these had not worked. I decided to test digest with Bg1II/XhoI, but I only ran the digest for 1/2 an hr because I was so hungry. I ran these digests on a gel and got only singly cut vector. I'll have to run these digests again for a longer period of time. Kdoan 00:15, 7 August 2007 (EDT)

8/5/07

I moved the T7+hemA+hemB plate from the incubator to the fridge. Kdoan 00:15, 7 August 2007 (EDT)

8/6/07

I autoclaved a 96-well block and added 800uL of LB/Amp media into each well. Then I picked single colonies. I placed the block into the shaker upstairs. Kdoan 00:15, 7 August 2007 (EDT)

8/7/07

I retrieved the 96-well block and centrifuged it for ten minutes. When I looked at the pellets, they were less brown than the ones with T7+hemA alone. I knew something had gone wrong so I cleaned up the block and set up new digests for T7+hemA1, T7+hemA2, and 52+hemB to combine them again. I made more gels, then set up test digestions with BglII/XhoI for ABD1, ABD2, ABD3, ACBD1, and ACBD2. After two hours, I ran a gel for the T7+hemA1, T7+hemA2, and 52+hemB. All three got good bands and sizes so I gel extracted T7+hemA1 and 52+hemB, gel purified, ligated, transformed into DH10B cells and plated on LB/Amp. I'm also going to prepare for my assay and do a practice run on ShemA+B and ShemAC+B. So the first step is to do plasmid transformations of each miniprep into DH10B and plate on LB/Amp as well. I also plated a negative control of DH10B with water and KCM, but no plasmid on a plain LB plate. I then helped Arthur pour and flame plates. Kdoan 15:06, 7 August 2007 (EDT)

8/8/07

I checked out my four plates and all had ample colonies. I added 2mL of LB media to the (T7+hemA)+hemB in DH10B and scraped. I then extracted 70uL of the mixture and miniprepped this. Then I did a plasmid transformation in BLR cells on plated on an LB/Amp plate. I took 250mL flasks and added 100mL of LB media to one flask labeled DH10B (negative control) and 100mL of LB/Amp media to flasks labeled ShemA+ShemB in DH10B and ShemAC+ShemB in DH10B. I then picked one colony from each corresponding plate and grew in the correct flask. I then placed these flasks in the incushaker. For the rest of the day, I updated the wiki and registry with many many parts. Kdoan 13:39, 8 August 2007 (EDT)

8/9/07

I looked at my three 250mL flasks and noticed the DH10B negative control flask was clear. Maybe I didn't pick the colony or accidentally grew in LB/Amp media. So I plated more DH10B competent cells on an LB plate. The other two 250mL flasks containing ShemA+ShemB and ShemAC+ShemB were brownish and cloudy so I placed these flasks in the fridge. I looked at my T7+hemA+hemB in BLR and noticed only six colonies. So I performed a second plasmid transformation of my T7+hemA+hemB in DH10B miniprep into BLR cells and plated onto an LB/Amp plate. I also made more gels. Kdoan 13:54, 13 August 2007 (EDT)

8/9/07

Nhu took my DH10B negative control plate and T7+hemA+hemB in BLR plate and put them in the fridge. Thanks Nhu! Kdoan 13:54, 13 August 2007 (EDT)

8/12/07

I picked a colony from my second DH10B plate and grew it in 100mL of LB media in a 250mL flask. I placed the flask in the incushaker. I looked at my T7+hemA+hemB in BLR plate and this time, there were many colonies. I obtained two 96-well blocks from upstairs and added 1mL of LB/Amp media to each well. I picked colonies from the T7+hemA+hemB plate in BLR and grew one in each well. I tried to avoid the colonies that were white and picked the ones that were more beige-colored. I placed the gauze sticky over both and brought them upstairs to the special incushaker. Kdoan 13:54, 13 August 2007 (EDT)

8/13/07

I obtained my two 96-well blocks from upstairs and spun them down in the large centrifuge. I picked two from each block that belonged to the group of wells that were browner and darker than the rest. I grew these in LB/Amp culture and placed the four tubes in the incushaker. I kept my 96-well blocks in the fridge just in case. My DH10B negative control flask was cloudy this time so I gathered the three 250mL flasks and performed a UV-Vis to measure the absorbances. I spun down 5mL of each culture, flicked out the supernatant, resuspended in 500uL of EB buffer, added 300uL of P2 lysis buffer, 300uL of 0.5M HCl, centrifuged at 3750rpm for 10min, then extracted 1mL from each supernatant and transfered to labeled plastic cuvettes. I also had a blank with EB buffer, P2, and HCl. I took these cuvettes to the Beckman machine upstairs, set the wavelength range to 250-800nm, and measured the absorbance for the blank first. Then, I measured the DH10B, ShemA+ShemB, and ShemAC+ShemB. The computer plotted the lines on one graph and I printed out the data. I then completed updating the registry and my notebook. Kdoan 14:02, 13 August 2007 (EDT)

8/14/07

I miniprepped T7AB1-4 and ran test digestions with Bg1II/XhoI. After an hour, I ran a gel with these samples and all four got the correct sized bands! I planned out my digestion for tomorrow and asked around about the LCMS. I also prepared a little for the presentation on Thurs. Kdoan 14:50, 14 August 2007 (EDT)

8/15/07

I digested T7AB1-3 and 51+hemC with the appropriate enzymes to combine them. I did multiple T7AB and two 51+hemC digestions to ensure at least one works. I made more gels and prepared for the presentation. I also helped David with autoclaving. After two hours, I ran the digestions on a gel. I got the right sized bands for all except T7AB2, but T7AB1 and T7AB3 were good. I gel extracted and combined the two 51+hemC into one tube. I gel purified, ligated T7AB1 and T7AB3 with 51+hemC, transformed into DH10B, and plated on LB/Amp plates. Kdoan 17:15, 15 August 2007 (EDT)

@@ Line 3: / Line 3: @@
 [[Template:BerkiGEM2007_KristinSequencingFiles | My Sequencing Files]]<br>
 ----
-==~~!~~==
+==6/6/07==
-notes
+Today was a pretty easy day.  We began with trying to continue the project that was very much unsuccessful yesterday.  We wanted to make a composite part with the RFP insert downstream of the p-Tet promoter. We digested pBca9145-1144 to retrieve our RFP insert as well as pBca9145-9205, which contains the promoter. low and behold, we failed again. all the lanes with the RFP showed up beautifully, but no lanes showed up for the 9205 plasmid. BUT!! it may not be our fault because the 9205 DNA was never tested. so instead, we worked on our wikis. The day wasn't in total vain because I did read two articles on heme.
-=== ===
+[[User:Kdoan|Kdoan]] 17:37, 6 June 2007 (EDT)
+==6/7/07==
+We began by setting up PCRs with the new oligos we received.  but there were only one set of pipetmans. since we're a team, we made do. I was overwhelmed by six PCR reaction tubes.  One for hemB, one for hemD, and two each for hemA from R. Capsulatus and hemC since they both have internal restriction sites. After they completed, I ran the two PCRs for hemA and two PCRs for hemC on a gel to gel purify.  Horrible result: only one PCR reaction from hemA and one PCR reaction from hemC worked. The ones that worked were the ones with oligos kd003/kd002 and kd009/kd008.  As a result, I extracted/dissolved the gels of the PCR reactions that worked and dissolved in ADB to store in the fridge.  I can't proceed with two good PCR products without the other two.  I then set up new reactions to redo my failed PCRs.  One for the failed hemA with oligos kd001/kd004 and one for the failed hemC with oligos kd007kd010 and ran these PCRs on the machines upstairs.  Inbetween all of this, I ran an analytical gel to see my PCRs from hemB and hemD.  Good news: they worked! I saw bands corresponding to 1006bp of hemB and another to 772bp of hemD. I then took a picture of this gel and cleaned up the PCR product. Afterwards, I digested with Bg1II/XhoI/DpnI, ligated with the vector pBca9145-1144#5 that Austin has prepared and digested for us, and then transformed into DH110B competent cells.  I made one plate for hemB and another for hemD. Quite a busy series of events! [[User:Kdoan|Kdoan]] 19:20, 8 June 2007 (EDT)
 <span style='font-family:"Courier New";color:#3366FF;font-size:6.0pt'>
 to do
 </span>
+==6/8/07==
+nd day: things were in a little more control today. There were colonies for both plates, white ones! I gave them more time to grow.  I set up two PCR tubes for hemA from CFT073 E.Coli.  Two again because there's an internal restriction site. One had oligos kd013/kd016 and another had kd015/kd014. I ran those and during this time, I received my PCRs that I had left upstairs from the day before.  The PCRs that I had to redo.  I ran them on a gel to gel purify and this time there were bands. I excised a 639bp band from hemA R. capsulatus and a 306kb band from hemC and dissolved in ADB buffer.  I retrieved the two PCR reactions for hemA and hemC that I had refrigerated the day before and combined the two hemA products with the two hemC products.  I cleaned up the gel and isolated the DNA and eluted with 15uL of water.  I then ran one PCR reaction to ligate the two PCR products of hemA with kd001/kd002 and another to ligate hemC with kd007/kd008.  After I ran this PCR and it completed I refrigerated the products to test them out on Monday.  When the colonies of my plates had grown to adequate size, I selected four from each that I would run colony PCRs on.  I labeled the ones from hemB B1-B4 and the ones from hemD D1-D4.  I prepared master solutions to perform four PCRs for hemB and four for hemD.  I used oligos kd005/kd006 for hemB and kd011/kd012 for hemD again. I used pipette tips to extract cells and add as a template to each PCR tube.  Afterwards, I would make an "X" of cells on a fresh plate. I labeled the colonies I picked 1-4, the PCRs 1-4, and my new plate had 1-4 in each corner. I then ran a PCR with the eight rxn tubes and incubated my new hemB and hemD plates in the 37C.  Austin will check on them tomorrow and move the plates to the 4C fridge. Before I left, I ran a test gel to see if my two PCR reactions for hemA from CFT073 worked.  And they didnt! I'll have to set those up again on Monday.  [[User:Kdoan|Kdoan]] 17:16, 11 June 2007 (EDT)
+==6/11/07==
+I started off today with running analytical gels to check if my PCRs from Friday worked.  I ran one gel to see if my eight colony PCRs worked. Bad news: no bands appeared! However, I highly doubt that my colonies do not have the insert because none of them turned red.  Farnazz and Amin said that their colony PCRs also often fail so I decided to move on and grow my four colonies with hemB and four colonies with hemD in culture anyway.  I scraped colonies from the new plates with the "X" of cells and placed them in the shaker to incubate overnight.  I ran another test gel to see my results for the other PCR reactions. The one PCR to combine the hemA from R.cap and the other PCR to combine hemC.  This PCR also failed. So I set up two more PCR reactions.  One to combine the hemA from R.cap and another to combine hemC and ran these upstairs.  I then poured my agarose gels. Then I set up two more PCRs to amplify the hemA from CFT073. When I ran these gels, all my PCRs had failed.  This was a bad day! I'll have to see what I'm doing wrong... [[User:Kdoan|Kdoan]] 17:29, 11 June 2007 (EDT)
+==6/12/07==
+So I realized that I've been adding 1ul of PCR buffer instead of 5ul. This is not good because without the buffer, the  DNA polymerase degrades and nothing gets amplified.  So I set up yet another PCR reaction to combine the two products of hemA and two products of hemC.  While I waited, I obtained the eight cultures I grew overnight and miniprepped the four cultures of hemB and four of hemD.  In order to see if my plasmids are correct and if they could possibly contain the right gene I ran test digests.  I digested 5uL from each miniprep with Bg1II and Xho1 then ran all eight onto a gel.  Good thing: for each lane, I observed one large band corresponding to the plasmid and a smaller band, which is most likely the gene.  I looked at the sizes and the largest band was approximately 2000bp, which is the size of the plasmid.  The smaller band for the hemB minipreps was around 1000bp, which is consistent and the smaller band for hemD was around 800bp, which is also good.  I then picked two samples from each gene: B2, B4, D2, and D4.  I sent kd001hemB2, kd002hemB4, kd003hemD2, kd004hemD4 for sequencing at Quintara.  I checked my PCRs for hemA and hemC and this time I got bands! So I cleaned up the PCR products, digested with Bg1II/Xho1/Dpn1, cleaned up the digest, ligated with the vector pBca9145-1144#5, and then transformed into DH110B competent cells. I made one plate for hemA and another for hemC. [[User:Kdoan|Kdoan]] 17:50, 12 June 2007 (EDT)
+==6/13/07==
+I set up more PCRs to redo my hemA from CFT073.  This hemA also has an internal restriction site so I had to divide into two PCRs.  However, my CFT073 had run out! So I tried my best by adding 1uL of water and centrifuging hoping to collect any CFT073 on the sides.  When I ran the PCR results on a gel, only a band showed up for the kd013/kd016 and not the kd015/kd014.  So I gel extracted and dissolved the band I did see in ADB buffer and refrigerated until I get more CFT073.   I then looked at my hemA and hemC plates and chose three colonies from each (hemA1-hemA-3 and hemC1-hemC3) and grew them in culture.  I then analyzed my sequencing results for B2, B4, D2, and D4.  D2 was short so I immediately threw that out.  The first 600-650bp of B4 and D4 were good but the sequences of hemB and hemD are 1006bp and 772bp, respectively.  So I sent in another 8uL of my minipreps and included G01001, the reverse primer, to get the end of my gene so I could analyze the last few hundred bp and check for mutations.  [[User:Kdoan|Kdoan]] 18:26, 14 June 2007 (EDT)
+==6/14/07==
+I obtained the cultures that I had grown overnight and miniprepped the cultures from colonies hemA1-hemA3 and hemC1-hemC3.  I then took 5ul from each miniprep and digested with Bg1II and Xho1 to see if this digestion would separate my plasmid into parent vector and insert.  When I did this and tested on a gel, I saw my gel had been smeared strangely.  It looked as if my A1 and A2 were squished together, while the others were ok.  Just in case, I chose A3 and C1 to send for sequencing.  This time, I sent in two samples of each miniprep and asked quintara to sequence with both ca998 and G01001 so I can analyze the entire sequence of my gene.  Afterwards, I analyzed the sequencing results of B4 and D4 with G01001.  After some struggling, I confirmed that the last 600-700bp matched up using the new sequencing results. Even though the first 100 or so were a little off with these sequencing results because the primer can only go for so long, I knew my B4 and D4 were fine because the sequencing results of the day before showed the beginning 600-650 bp are good. [[User:Kdoan|Kdoan]] 18:20, 28 June 2007 (EDT)
+==6/15/07==
+I began with looking at my sequencing results for A3 and C1.  Both had many mutations when compared to the predicted PCR products.  I discarded these results assuming they were faulty and sent in A2 and C2 to see if these clones were matches.  Again, I sent in two samples of each and included ca998 and G01001 because these genes are too large for just the reverse.  I then began the process of adding the RBS to hemB4 and hemD4.  I made construction files for the composite part RBS + hemX and obtained the RBS.  I digested the RBS with BamH1 and XhoI and the heme genes with Bg1II and XhoI.  I ran my digestions on a gel and excised the bands.  However, at this point, I discovered I was given the wrong RBS.  I was given a pooled sample of RBS, while what I needed is pBca1101-I716051-kan cassette.  So I drew up new construction files for the correct RBS composite part. I was able to keep the gel bands for hemB and hemD, but I had to re-digest the new RBS I received with BamHI and XhoI.  After I waited for the digest and excised the bands for the rbs, I purified the two RBS, hemB, and hemD digestion products. [[User:Kdoan|Kdoan]] 18:23, 28 June 2007 (EDT)
+==6/17/05==
+Yes, I came in on a Sunday.  No one else was here except for John for an hour.  During my stay, I began the cloning process for hemA from CFT073.  All my previous attempts had failed because I was adding 1uL of buffer instead of 5uL.  I obtained more CFT073 from Chris.  This hemA from CFT073 also has an internal restriction sites so my first PCR reactions were to eliminate this Bg1II site.  One PCR reaction included oligos kd013/kd016 and another included kd015/kd014.  Afterwards, I ran my PCR products on a gel, excised the bands, and purified the gel bands.  I  then ran a second PCR to combine these two fragments to get the entire hemA gene from CFT073 and left the PCR tube in the machine overnight.  I then resumed with the making of the composite part of RBS+hemB and RBS+hemD.  I had digested these, ran them on gels, excised the bands, and cleaned up the gel bands.  I then ligated RBS+hemB and RBS+hemD and transformed in DH110B competent cells.  Because this RBS has a kan cassette and not a carb, I had to add 100uL of LB media and put the transformation reactions in the incushaker for one hour for the cells to start production of kanR so they wouldn't die immediately on the LB+kan plates.  I then plated each transformation reaction on two plates and left them overnight to grow. [[User:Kdoan|Kdoan]] 18:23, 28 June 2007 (EDT)
+==6/18/07==
+My two plates of RBS (pBca1101-I716051-kan cassette) + hemB and RBS + hemD had colonies so I chose three from each (hemB+rbs1-3 and hemD+rbs1-3 and grew them in LB+carb media.  It would've been better if I grew these colonies in LB+kan, but the RBS I used also has an AmpR gene so my cells will still live.  I worked around a flame so there should be little chance of contamination.  Afterwards, I obtained the PCR reaction I left overnight of hemA from CFT073, ran an analytical gel and saw a product.  I then cleaned the PCR product, digested with Bg1II/XhoI/DpnI, ligated with pBca9145-1144#5 vector, and transformed into DH110B competent cells.  I then plated and left it in the 37C incubator overnight.  I then proceeded with looking at the sequencing results I received over the weekend. Both A2 and C2, like A3 and C1, had mutations.  However, upon close inspection, I noticed that the mutations of C1 and C2 were identical.  Two different GC-->CG mutations in the same position. Otherwise, these clones were fine.  I looked up the sequence for hemC I found on colibase and compared them to pubmed.  These differences matched the differences  I noticed with my clones.  My clones matched perfectly with the ones in pubmed.  So both my C2 and C1's worked.  I then turned to A2 and A3.  A3 had many mutations.  A2 also had a GC-->CG mutation which matched with two of the many mutations I saw in A3.  I couldn't find another hemA R.capsulatus sequence that could verify that I was working with a different template from the one displayed in pubmed.   I consulted Chris and he said with 90% assurance that the mutations were OK to work with since both clones had the same mutations.  Also, I asked Richard from Quintara to redo my A2 and C2 with G01001 because they sequenced with ca998 and I need to analyze the second half of my gene. [[User:Kdoan|Kdoan]] 18:21, 18 June 2007 (EDT)
+==6/19/07==
+After growing my hemB+rbs1-3 and hemD+rbs1-3 in culture, I miniprepped all three and began to run them on test gels to make sure that when I cut with BglII and XhoI I would see one band corresponding to insert and one band corresponding to parent vector.  However, Chris then informed me that I had done them incorrectly.  Instead of cutting out the RBS and putting it in front of the heme gene, I cut out the heme gene and put it behind the RBS.  So even though the two were in the correct order, the parent vector pBca9145 would not be maintained.  So I decided to just wait until I had all five of my heme gene basic parts ready (hemA from CFT073, hem A from R.capsulatus, hemB, hemC, and hemD) and add the RBSs all at once.  I then also learned that I had to add two different kinds of RBS to each heme gene.  One RBS library to each and one single RBS to each.  I chose pBca1101-1106B as the single RBS to add to each heme gene.  As for the RBS library, I need to alternate between pBca1101-I716051, which has a kan cassette, and pBca1101-I716052, which has a cam cassette.  So hemA from CFT073, hemA from R.capsulatus, and hemC will get the RBS library with the kan cassette and hemB and hemD will get the RBS library with cam casette.  I planned out exactly how I was going to do this.  Afterwards, I chose three colonies from the hemA from CFT073 plate and grew these in culture.  Then I analyzed my sequencing results.  A2 and C2 with G01001 and ca998 looked great and these are the colonies that I will add RBS's to. [[User:Kdoan|Kdoan]] 18:22, 28 June 2007 (EDT)
+==6/20/07==
+I decided to postpone the intense RBS adding until I had all five of genes sequenced and ready to go.  So I miniprepped the three colonies from hemA from CFT073, digested 5uL from hemAC1, hemAC2, and hemAC3 with BglII and XhoI, and ran these samples on gel.  They all looked good so I chose to send hemAC1 and hemAC2 for forward sequencing with ca998 and reverse with G01001.  Austin told me we had few pBca1101-I716052 and pBca1101-I716o51 left, so he handed me one culture of each and I miniprepped 50uL more of each.  Since I had a free day afterwards I made more gels and helped set up some shelfs for the new fridge.  I then transferred some bottles and plates from the old frigde to the new one.  I checked over my plans and made sure I was ready to go for the next day. [[User:Kdoan|Kdoan]] 18:21, 28 June 2007 (EDT)
+==6/21/07==
+Long day. I had 13 digestions going on.  Five for the heme genes being cut with EcoR1/BglII to add to the RBS libraries, five for the heme genes being cut with BglII/XhoI to add to the single RBS, and three for each kind of RBS (one for I716051, one for I716052, one for 1106B).  I digested for an hour.  Ran all of them on gels.  Extracted the bands and gel purified.  Afterwards, I ligated with their appropriate pairs and transformed into DH110B competent cells.  The five being added to the single RBS 1106B I was able to plate right away on LB-Amp plates, but the other ones I had to add 100uL of LB media and grow in the incushaker for about 30 minutes.  Afterwards, I plated  hemA from R.capsulatus, hemA from CFT073, and hemC on LB/Amp/kan plates because these were ligated to the RBS I716051 with kan resistance while hemB and hemD were grown on LB/Amp/Cam plates because these were ligated to the RBSI716052 with cam resistance.  I grew these in the 37C incubator overnight. [[User:Kdoan|Kdoan]] 18:22, 28 June 2007 (EDT)
+==6/22/07==
+Lab got flooded today! Disaster! People moved our plates from the 37C incubator to the fridge.  I came in for a bit to check out the situation.  I also looked at my plates and all had colonies. [[User:Kdoan|Kdoan]] 18:21, 28 June 2007 (EDT)
+==6/25/07==
+I chose two colonies from each plate. So I had twenty tubes.  The 1106B RBS's I was able to grow in just LB-Amp culture, but the I716051 RBS's I grew in LB-Amp-Kan and the I716052 RBS's I grew in LB-Amp-Cam. [[User:Kdoan|Kdoan]] 18:21, 28 June 2007 (EDT)
+==6/26/07==
+I discovered that for the I716051 and I716052 RBS libraries I'm not supposed to just pick single colonies and grow these in culture.  I'm supposed to add 2mL of LB media to each plate, spread until the mixture is homogenous, extract 1uL of mixture and grow this 1uL in the appropriate LB-antibiotic media.  So I threw away the ten cultures I grew up overnight that corresponded to single colonies I picked from the gene+RBS library plates.  Then I grew up the RBS library colonies the proper way in five culture tubes with the appropriate LB-antibiotic media. I retrieved my ten culture tubes and began to miniprep.  I noticed that almost all of them had a pinkish tint, but didn't look into it.  When I transferred some liquid to centrifuge tubes and pelleted the cells, I noticed the cells were pink.  I knew that this was from RFP.  When I looked at my construction files, I saw that if my digestions had worked properly, the RFP should've been cut out. I consulted Amin, and he said that my digestions were probably inadequate and next time I should wait for at least two hours.  Chris checked my construction files and everything should work ok.  I noticed there wasn't enough 1106B RBS so I decided to use 1106A so I changed my construction files.  I prepared for the next day and made more agarose gels. [[User:Kdoan|Kdoan]] 18:20, 28 June 2007 (EDT)
+==6/27/07==
+This time I ran my digestions for two hours. I ran them all on gels.  However, rbs I716051, 1106A, hemC digested with EcoR1/BamH1 and hemD cut with Bg1II/XhoI were still inadequately cut.  Chris suggested I mix more thoroughly. I  extracted the other bands and melted them in ADB buffer and refrigerated.  I set up the digestions that didn't work as well and during this time, we had our meeting.  Afterwards, I ran the re-digestions on a gel, gel extracted, and purified all thirteen digestions products and eluted each with 10uL of water.  Now they're ready for ligations tomorrow. [[User:Kdoan|Kdoan]] 18:20, 28 June 2007 (EDT)
+==6/28/07==
+I ligated the appropriate RBS with its corresponding heme gene.  I then transformed into DH110B competent cells. The ones ligated to 1106A RBS I plated immediately on LB-Amp plates, but the RBS libraries I grew in 100uL of media for one hour then plated them onto their appropriate plates.  I made more gels and updated the registry with my parts.  I also noticed that my hemB4 and hemD4 were running low and just in case my RBS additions failed again, I would need more of these minipreps.  So I picked more of these colonies and grew them in culture once more so tomorrow I could miniprep. I also helped out Sam by gel purifying two of her samples and David with autoclaving tips. [[User:Kdoan|Kdoan]] 18:20, 28 June 2007 (EDT)
+==6/29/07==
+I retrieved my ten plates. I need to wait longer before the colonies with parent vector turn pinkish-red from RFP.  I miniprepped the hemB4 and hemD4.  I picked colonies from Vai's plates and grew them in 12 tubes with LB-Amp culture.  I also made more LB-Carb media because the stock was running low.  I then chose colonies to grow in culture from the ten plates.  Well, for the five RBS libraries, I added 2mL of LB media, spread this around, then grew 1uL of the mixture in the appropriate LB+antibiotic media. For the other five plates with the single RBS 1106A, I chose two colonies from each.  I looked at the plates under the UV lamp to make sure the colonies I chose did not glow pinkish from the RFP gene.  I then grew these clones in LB-Amp media. [[User:Kdoan|Kdoan]] 15:15, 29 June 2007 (EDT)
+==6/30/07==
+I came in on a Saturday! I retrieved my fifteen culture tubes from the incushaker.  Upon initial inspection, I noticed that 1106A+hemC1 was the only one that looked pink, most likely these DH110B cells had taken up uncut 1106A parent vector, which also has the RFP gene.  The 1106A+hemA1 and 1106A+hemA2 looked quite brownish and the 1106A+hemAC1 and 1106A+hemAC2 was a lighter brown.  These browns were distinctly different from the pink from the RFP.  I pelleted the cells and as I suspected, the 1106A+hemC1 was pink from the RFP and it fluoresced when viewed under UV light.  The 1106A+hemA1/2 and 1106A+hemAC1/2 were brownish (1106A+hemA1/2 was darker brown)and Chris and I believe that this is from the production of heme.  Heme has a brownish/red phenotype so hopefully this brown pellet is from the accumulation of heme. I then completed the 15 minipreps.  Afterwards, I moved Vai's culture tube and two transformation plates from the incubator room to the fridge. [[User:Kdoan|Kdoan]] 14:55, 2 July 2007 (EDT)
+==7/2/07==
+I do not need to send the RBS libraries in for sequencing, but the single RBS 1106A I will send for sequencing.  Even though the nine 1106A+gene clones did not become pink and so they do not contain uncut RBS parent vector, they can still contain uncut pBca9145-gene parent vector.  So I ran a test digest where I cut these minipreps with Bg1II/XhoI and ran the nine minipreps on a gel. However, I noticed this lane of gels I made the wells cut all the way through the bottom.  So my gels were too thin for how low the combs were. I checked to make sure the other gel lanes didn't have this problem.  Some of my samples ran out but I hoped there was enough DNA to detect.  The ones that didn't run out had the correct size bands but there were three lanes (hemA2, hemAC1, hemAC2), which had nothing appear and coincidentally, these were the same ones where my DNA samples had run out. I decided to send one from each gene+1106A RBS sample anyway for sequencing. I sent hemA1, hemAC2, hemB2, hemC2, and hemD2 for forward sequencing with ca998. I then made construction files to link together hemA+hemB, hemAC+hemB, and hemC+hemD for both the single and library RBS. [[User:Kdoan|Kdoan]] 14:55, 2 July 2007 (EDT)
+==7/3/07==
+I looked at my sequencing results.  RBS+ gene for HemA1, hemAC2 hemB2, and hemD2 were good except they had two identical mutations.  These came before the RBS+gene and after any restriction sites so I assumed these were ok. HemC2 Richard said did not work, but I proceeded anyway.  I followed my construction files to set up the ten digests.  Five from the libraries and five from the single RBSs.  After I put the digests in the 37C, I realized that for the libraries, the way in which I digested would only keep the kanR gene from hemA, hemAC, and hemC, but cut out the CamR gene from hemB and hemD.  Chris had told me to alternate antibiotic resistance genes and I figure that would be for a reason.  So I emailed him to ask about this and decided to wait on the libraries until I know exactly what I am supposed to do.  After two hours, I ran my digests on a gel, but did not get bands for hemC2 cut with BamHI and XhoI.  So I redid this digest and cut out the other ones and gel purified.  After another two hours, I ran hemC2 again on a gel but still did not get bands.  Maybe I did my miniprep wrong and that is why quintara had difficulty getting sequencing results.  I still had the digested hemC with Bg1II/XhoI and RBS 1106A with BamHI/XhoI so I decided to re-ligate and transform this since my 1106A+hemC plate had only 7 colonies and only one was white. So I did this transformation along with the hemA+hemB and hemAC+hemB.
+[[User:Kdoan|Kdoan]] 14:55, 2 July 2007 (EDT)
+==7/4/07==
+I looked at my plates. For some reason, hemA+hemB did not have any colonies.  I still have the digested/purified hemA as well as the digested/purified hemB, so I will just re-ligate and transform tomorrow.  I chose two colonies from the 1106A+hemC and hemAC+hemB plates and grew these in LB+Amp media. [[User:Kdoan|Kdoan]] 17:01, 5 July 2007 (EDT)
+==7/5/07==
+The two colonies I chose from 1106A+hemC turned pink from the RFP.  I looked at the original plate and all the colonies were pink.  I threw this plate away.  I can't use the digested/purified hemC because this contains too much undigested parent vector. I'll have to re-digest hemC2 tomorrow and ligate this to 1106A RBS.  I digested the RBS libraries+gene to make composite parts (hemA+hemB, hemAC+hemB, and hemC+hemD).  When hemA is cut with EcoR1/BamHI/ScaI, there are 1.5kb and 1.27kb bands which will be hard to resolve so I'll need to run the gel slow and add EtBr to the buffer. After 2-hr digestions, I ran the samples on a gel and gel extracted, but I didn't get bands for hemA. I gel extracted and purified the others and ligated them.  I then transformed into Dh110B cells and then added 100uL of LB media to each transformation and incubated for 30 minutes because I'll be plating on LB+Amp+Kan plates.  I then plated the hemAC+hemB and hemC+hemD transformations. During the two hour digest, I also miniprepped the two colonies I picked from hemAC+hemB (single 1106A RBS), made more gels, and re-ligated and retransformed the single RBS hemA+hemB since I didn't get any colonies the first time.  The two minipreps I digested  5uL with Bg1II/XhoI to see if I would get the correct bands assuming my cassette is kanR-RBS-hemAC-RBS-hemB. I then ran these on a gel, but my bands were very faint so I ended up sending both for forward and reverse sequencing anyway.
+[[User:Kdoan|Kdoan]] 14:55, 2 July 2007 (EDT)
+==7/6/07==
+Both the minipreps for single RBS hemAC+hemB for forward and reverse sequencing were good.  I set up digests for single RBS hemA and library RBS hemA to string them together with the single RBS hemB and library RBS hemB that I already have purified/digested.  Unfortunately, the plates I did for library RBS hemAC+hemB and library RBS hemC+hemD did not yield enough colonies for a library.  They did get maybe 20 colonies for each plate. Because I still have the purified/digested products for library RBS hemX, I just need to re-ligate and re-transform. I did plasmid transformations for hemA2, hemC2, Bca1101-1106A, and Bca1101-1106B because these minipreps are running low. I also made more gels. After single RBS hemA and library RBS hemA digested for two hours, I ran these samples on a gel and extracted the appropriate bands.  I cleaned up the digest, ligated, and transformed into DH110B cells.  I plated the single RBS hemA+hemB right away but the other ones I incubated in 100uL of LB and plated on LB+Amp+Kan plates.
+[[User:Kdoan|Kdoan]] 14:55, 2 July 2007 (EDT)
+==7/7/07==
+I came in really quickly to grow things in culture.  For the ShemA+ShemB plate, there was only one colony.  Chances are it won't be right, but I decided to grow this one colony in culture anyway.  The LhemAC+LhemB plate had only about 15 colonies so I'll have to redo this one.  The LhemA+LhemB had 30 colonies so I added 2mL of LB, spread the media around, then grew 1uL in LB+Amp+Kan media.  The 1106A, 1106B, hemA2, and hemC2 plates had plenty of colonies.  The 1106A/B plates had pink colonies as expected.  So I just chose one colony from each and grew these in LB+Amp media. [[User:Kdoan|Kdoan]] 17:25, 9 July 2007 (EDT)
+==7/8/07==
+I retrieved my cultures from the incushaker and miniprepped all seven tubes.  The minipreps for 1106A/B I put back into PB#3.  The hemA2, hemC2, ShemA+ShemB1 and LhemA+LhemB minipreps I put in my miniprep box and I sent ShemA+ShemB1 for forward and reverse sequencing.  [[User:Kdoan|Kdoan]] 17:25, 9 July 2007 (EDT)
+==7/9/07==
+I digested hemC2 in order to ligate it to single RBS 1106A.  I also digested 51+hemAC in order to make the composite part (RBS library) hemAC+hemB.  I made gels during this time.  After two hours, I ran the two samples on a gel, extracted the appropriate sized bands, and purified.  I then ligated hemC2 to single RBS 1106A and 51+hemAC to 52+hemB.  Single RBS 1106A and 52+hemB were previously digested and purified.  I transformed into DH110B cells and plated hemC2+1106A on LB+Amp plates.  LhemAC+LhemB I had to incubate for one hour in 100uL of LB media because I plated this transformation on LB+Amp+Kan plates.  [[User:Kdoan|Kdoan]] 17:43, 9 July 2007 (EDT)
+==7/10/07==
+The plates I transformed were no good. The LhemAC+LhemB had only about 10 colonies, which is definitely not enough.  The 1106A+hemC2 plate had four colonies that were already turning pink.  I checked my sequencing result for ShemA+ShemB1, and the sequence contained only RBS+hemA and not RBS+hemB.  Chris suggested I use 4uL of each ligation product instead of one and transform into TG1 cells instead of DH110B cells. Since I still had the purified/digested products required for each ligation, I just religated and transformed. The 1106A+hemC2 and ShemA+ShemB I plated on LB/Amp plates, but the LhemAC+LhemB I rescued and plated on LB+Amp+Kan plates. I digested LhemA+LhemB and LhemC+LhemD to make the composite part hemA+hemB+hemC+hemD.  I also digested these composite part libraries to check to see if I would get the correct band sizes. After a two hour digestion, I ran an analytical gel and got the right band sizes for LhemA+LhemB and LhemC+LhemD.  However, my other gel containing the digestions to make my super composite part failed. I got more bands than I expected for each digestion, showing that I had incomplete digests. I will try the whole procedure again tomorrow.
+[[User:Kdoan|Kdoan]] 17:56, 10 July 2007 (EDT)
+==7/11/07==
+The ShemA+ShemB plate had four colonies. The LhemAC+LhemB had a lawn. 1106A+hemC2 had many colonies, most of which were pink, but about ten were white. I added 2mL of LB to LhemAC+LhemB, spread, and grew 1uL in LB+Amp+Kan culture. I chose all four ShemA+ShemB and four of the ten white colonies from 1106A+hemC2 and grew these in LB+Amp.  I digested mini LhemA+LhemB and LhemC+LhemD to string these composite parts together. I made gels during the two hour digestion. I ran the digestions on a gel and saw the same results as yesterday. In addition to the bands I expected for both samples, for LhemA+LhemB I got a smear of DNA below the smallest band. Amin says this must be uncut vector DNA.  Luckily, I needed the top band, which seemed to correspond to the 3.3kb band I wanted. The LhemC+LhemD got the two bands I expected as well as two other ones. I cut the largest band, which also seemed to correspond to the 3.8kb band I wanted. I cleaned these up, ligated them (4uL of each ligation product again), and transformed into TG1 cells. I rescued for one hour then plated on a LB+Amp+Kan plate.
+==7/12/07==
+I came in at 12:30AM and grew the LhemA+B+C+D in LB/Amp/Kan culture. When I came in later, I miniprepped the samples I grew in culture yesterday. The pellets of LhemAC+LhemB were pretty brown and so were ShemA+ShemB1 and 4.  I decided to send 1106A+hemC3/4 for sequencing because these were slightly brown compared to the other two. I set up digests to combine LhemAC+LhemB and LhemC+LhemD. I also digested the ShemA+ShemB1-4 and LhemAC+LhemB with BglII/XhoI to see if I got the correct sized bands. I ran a gel and extracted the bands. Again, I saw four bands (???) for LhemC+LhemD and extracted the 3.8kb band. For the test digests, ShemA+ShemB4 looked the best and LhemAC+LhemB had the band sizes I expected. I gel purified LhemAC+LhemB and LhemC+LhemD, ligated them with 4uL of each, transformed into TG1 cells, rescued for one hour, then plated on an LB/Amp/Kan plate. During this time, I sent kd031-kd034 for sequencing and miniprepped LhemA+B+C+D and ran a test digest w/ BglII/XhoI. I expected a 4kb and 3kb band, but got 3 and 2kb bands instead. I suspect this might be because I cut the wrong band for the LhemC+LhemD digest, which mysteriously gave me four bands three times. Maybe I extracted uncut LhemC+LhemD plasmid. I'll try again. I suspected something was wrong because the pellet of LhemA+B+C+D was not brown. [[User:Kdoan|Kdoan]] 18:30, 12 July 2007 (EDT)
+==7/13/07==
+I grew the LhemAC+B+C+D in LB/Amp/Kan culture even though I think it's wrong since the LhemC+LhemD got four bands and I extracted the top band again. The same thing as the LhemA+B+C+D will probably happen so just in case, I redid both LhemA+B+C+D and LhemAC+B+C+D. This time, LhemC+LhemD had two bands, and the correct ones! So I gel extracted and purified. I ligated, transformed into TG1 cells, rescued for one hour, then plated on a LB/Amp/Kan plate. I also made gels and helped David with autoclaving tips. Quintara did not give me results for 1106A+hemC3/C4 so I digested both. Only 1106A+hemC4 gave me the correct band sizes so I ligated this product to ShemD, which I had purified/digested previously and transformed into TG1 cells again and plated immediately on a LB/Amp plate. [[User:Kdoan|Kdoan]] 13:14, 16 July 2007 (EDT)
+==7/14/07==
+I came in briefly to pick four colonies from the ShemC+ShemD plate and grow these in LB/Amp culture. I also grew 1uL of both LhemAC+B+C+D2 and LhemA+B+C+D in LB/Amp/Kan culture. I moved the culture of LhemAC+B+C+D1 that I grew overnight on Fri to the fridge so I can miniprep all my cultures at once on Sun. [[User:Kdoan|Kdoan]] 13:17, 16 July 2007 (EDT)
+==7/15/07==
+I miniprepped all eight culture tubes. I noticed that some of my libraries did not have brown pellets at all and the ones that did were a light brown color. I'll run a test digest with Bg1II/XhoI on Mon and I'm prepared to have to do the process again. [[User:Kdoan|Kdoan]] 13:20, 16 July 2007 (EDT)
+==7/16/07==
+I ran test digests of LhemA+B+C+D and LhemAC+B+C+D1/2.  These should give me fragments of 4 and 3kb. In conjunction with these test digests, I ran digests of LhemA+B, LhemAC+B, and LhemC+D so I can begin stringing the genes together again just in case the test digests show me bad results. I made more gels and after two hours, I ran my samples on a gel. My test digests showed that the 4 and 3kb bands were there, but in addition to these correct bands, the LhemA+B+C+D had an extra band below (possibly uncut vector) and the two LhemAC+B+C+D samples also had two extra bands. I figured these extra bands are background since I am working with a library and at least the bands I want are there. I decided to ligate and transform the digests I ran and I'll just have multiple LhemA+B+C+D and LhemAC+B+C+D copies to assay. I extracted the bands from the new digests, purified, ligated, transformed into TG1 cells, rescued for one hour, and plated on LB/Amp/Kan plates.  [[User:Kdoan|Kdoan]] 16:44, 16 July 2007 (EDT)
+==7/17/07==
+Now that I have my LhemA+B+C+D and LhemAC+B+C+D parts, I now have to add David's T7 promoter in front. I digested LhemA+B+C+D and LhemAC+B+C+D1/2 with EcoR1/Bg1II for two hours and ran these on a gel. The LhemA+B+C+D got 4 and 1kb bands even though I was expected 6 and 1kb bands. The LhemAC+B+C+D1/2 got the bands I expected plus two other bands below the 6kb band. So I gel extracted LhemAC+B+C+D1/2 and gel purified, but I also repeated the same digests for all three samples for two hours. When I ran a gel, I got the same result. So my LhemA+B+C+D is no good and I gel extracted the 6kb band again from the LhemAC+B+C+D1/2. I purified these and ligated my products from the first digest with the T7 wildtype promoter. I transformed into TG1 cells and plated onto LB/Amp plates. [[User:Kdoan|Kdoan]] 16:35, 17 July 2007 (EDT)
+==7/18/07==
+I came in to pick three colonies from each of my LhemAC+B+C+D1/2 + T7 plates and grow in culture so that I can test if the T7 promoter as well as all four of my heme genes are there by PCR.  I then scraped the plate and extracted 70uL from each plate to miniprep and perform plasmid transformation into BLR cells. Along with these minipreps, I also miniprepped LhemAC+B+C+D3 and LhemA+B+C+D2.  I also cleaned up a digest for Vai and then we all went to the baseball game. [[User:Kdoan|Kdoan]] 15:50, 19 July 2007 (EDT)
+==7/19/07==
+Chris suggested that because my single RBS genes have been consistently more brown than the library RBS genes I should really concentrate more on the singles instead. In conjunction with this, I will backtrack to 51 library RBS+hemA, pick the clone that yields the brownest product and begin connecting hemB, hemC, and hemD from there. On the plus side, Chris told me that the single RBS 1106A already has a promoter on it. This explains why I've been getting brown products with the single RBS from the beginning. With regard to the single RBS's, I have hemA+hemB and hemAC+hemB, but I haven't been able to make the composite part 1106A+hemC. I've had multiple failed attempts so I digested 1106A and hemC, gel extracted, purified, ligated, transformed into DH10B cells, and plated on LB/Amp. I made gels during the two hour digest and performed plasmid transformation for 51+hemA and 51+hemAC into TG1 cells and plated on LB/Amp plates.  [[User:Kdoan|Kdoan]] 16:25, 19 July 2007 (EDT)
+==7/20/07==
+I went home to SJ for the weekend. I asked Austin to put my RBS 51 + hemA and RBS 51 + hemAC in the fridge and to select four non-pink colonies from the 1106A+hemC plate and grow them in LB/Amp culture. [[User:Kdoan|Kdoan]] 15:41, 23 July 2007 (EDT)
+==7/21/07==
+I asked Austin to take the four culture tubes (1106A+hemC1-4) from the incushaker and put these in the fridge. [[User:Kdoan|Kdoan]] 15:43, 23 July 2007 (EDT)
+==7/22/07==
+I came in today to make up for my absence on Fri. I looked at my four 1106A+hemC culture tubes and noticed two were pink from RFP.  I discarded these and kept the other two to miniprep on Mon. I decided to ligate and transform the remaining digested/purified single RBS 1106A and hemC2 so I could sent more for sequencing in case 1106A+hemC1 and 1106A+hemC4 do not work. There were 6uL of each digested/purified product left and I transformed into TG1 cells and plated on LB/Amp plates.  As for my plates RBS 51+hemA and RBS 51+hemAC, I decided to grow 96 clones in a 96-well block so I can select for single RBSs in the library that turn the cells most brown. I had to autoclave the two 96-well blocks. I put 1mL of LB/Amp media in each well and picked individual clones to grow in each well. Then I covered them with sticky gauze and put them in the special incushaker upstairs. [[User:Kdoan|Kdoan]] 15:48, 23 July 2007 (EDT)
+==7/23/07==
+I obtained the 96-well blocks from the incushaker and pelleted the cells. When I looked at the pellets, I noticed the clones from RBS 51+hemAC were browner than the ones from RBS 51+hemA.  Some were lighter than others, but many  were equally browner. So I just chose five of the browner ones from each plate, pipetted up and down to resuspend the cells, and extracted 1uL to grow in 5mL of LB/Carb/Kan media. I also grew one clone from an old hemC2 plate in 5mL of LB/Amp to act as a negative control so that tomorrow I can compare the brown w/ the non-brown and make sure there is a noticeable difference. I picked three clones (1106A+hemC2/3/5) from the new 1106A+hemC plate and grew these in LB/Amp culture and miniprepped the 1106A+hemC1/4 from before. I ran a test digest of 1106A+hemC1/4 with Bgl11/XhoI in parallel with a digest to begin combining these with 1106A+hemD, which I also set up a digest for.  Unfortunately, both 1106A+hemC1/4 had the wrong sized bands so I did not combine 1106A+hemC with hemD. So I redigested hemC1 clone and 1106A, ran a gel, got good bands, gel extracted, ligated, transformed into TG1 cells, then plated on an LB/Amp plate. [[User:Kdoan|Kdoan]] 16:00, 24 July 2007 (EDT)
+==7/24/07==
+I picked four colonies from the 1106A+hemC plates and grew these in LB/Amp culture. I compared the pellets from hemC2 with the five singles from 51+hemA and 51+hemAC. The 51+hemA and 51+hemAC were considerably darker. I picked one of each of the five singles and grew another 1uL in LB/Amp/Kan and I will compare these to 1106A+hemA/hemAC. Meanwhile, I miniprepped one single from 51+hemA and one from 51+hemAC.  I digested each with EcoR1/Bg1II, ran gels, gel extracted, purified, ligated these with T7 promoter that has already been digested with EcoR1/BamH1, transformed into TG1 cells, rescued for one hour, then plated on LB/Amp/Kan plates.  At the end of the day, I miniprepped the 1106A+hemC1-4 after only 5 hrs in the incushaker because I needed to send these in for sequencing asap.  Since the pellet was smaller I eluted in 40uL of water for the miniprep. [[User:Kdoan|Kdoan]] 15:51, 26 July 2007 (EDT)
+==7/25/07==
+I compared the pellets of 1106A+hemA/hemAC and 51+hemA and 51+hemAC. The 51+hemAC was darker than the 1106A+hemAC, while the 1106A+hemA is darker than the 51+hemA. It's not a fair comparison though since the 51+hemA/AC do not have promoters on them, while the 1106A has the Pcon promoter attached. I decided to go on with both experiments in conjunction. I added 2mL of LB to the T7+51hemA and T7+51hemAC plates, scraped, and extracted 70uL of mixture to miniprep. I miniprepped and did a plasmid transformation into 50uL of BLR cells and plated on LB/Amp/Kan plates. I checked my sequencing results. 1106A+hemC1/2 did not even have a ca998 binding site said Richard, 1106A+hemC3 had RBS then random sequence again, and 1106A+hemC4 was just hemC parent vector. I finally decided to try DBBS. I set up the two PCRs for DBBS. The first time, the PCR for 1106A worked, but hemC got no band. I set up a second PCR for hemC and left it overnight. I did the transformation of T7+51hemA/AC into BLR again just in case. [[User:Kdoan|Kdoan]] 16:16, 26 July 2007 (EDT)
+==7/26/07==
+For the second PCR reaction for hemC, I got two bands and none of them corresponded to the right size band. I set up yet another one for hemC, but at the same time, I transformed 1106A and hemC into lefty and righty strands so I can try John's method.  I autoclaved two 96-well blocks, let them cool for some time, added 1mL of LB/Amp/Kan media to each well, and picked colonies from my T7+hemA and T7+hemAC in BLR cells to grow in each well.  I brought the blocks upstairs to shake and left them overnight.  I made more gels. In the end, I checked my third hemC PCR by an analytical gel and this one yielded no bands again.  So I will move on to John's method. [[User:Kdoan|Kdoan]] 12:57, 30 July 2007 (EDT)
+==7/27/07==
+I realized I made the mistake of plating T7+hemA and T7+hemAC in both TG1 and BLR on LB/Amp/Kan plates. When I grew colonies in the 96-well blocks, I also grew on LB/Amp/Kan plates. So the clones I had been working with were uncut 51+hemA and 51+hemAC vectors because when cut with EcoR1 and Bg1II, the KanR gene is eliminated. I discarded all these plates and cleaned out the 96-well blocks. I began digesting 1106A hemA+hemB and 1106A hemAC+hemB and 1106A hemD so I can make this composite part for now and begin assaying by next week.  I will complete the part 1106A+hemC and make the full composite part asap. I couldn't find the miniprep for 1106A+hemD so I miniprepped from an old culture hoping that it's still good to work with. After two hours, the digestion 1106AhemA+hemB yielded three bands while the 1106AhemAC+hemB yielded no bands. I cut out the band that seemed correct from 1106AhemA+hemB and surprisingly, the 1106A+hemD yielded the two correct bands. I cleaned up, ligated, and transformed into TG1 cells and plated on LB/Amp.  At the same time, I ligated pre-digested/purified products of 51+hemA and 51+hemAC with the T7 promoter and transformed into TG1 and plated on LB/Amp plates this time. I picked one colony from each of the lefty 1106A and righty hemC plates and grew these clones in LB/Amp cultures. [[User:Kdoan|Kdoan]] 13:10, 30 July 2007 (EDT)
+==7/28/07==
+I added 2mL of LB media to the T7+hemA and T7+hemAC, scraped, and extracted 700uL to miniprep.  I miniprepped these two samples along with the lefty 1106A and righty hemC.  During the ten minute centrifuge with P1, P2, and N3, I picked eight colonies from the ABD plate and grew them in LB/Amp cultures.  After the minipreps, I transformed 2uL of T7+hemA and T7+hemAC into BLR cells and plated on LB/Amp plates.  [[User:Kdoan|Kdoan]] 12:57, 30 July 2007 (EDT)
+==7/29/07==
+I came in very briefly to parafilm my T7+hemA and T7+hemAC in BLR plates and move them to the fridge.  I also transformed my ABD1-8 cultures tubes to the fridge.  [[User:Kdoan|Kdoan]] 12:57, 30 July 2007 (EDT)
+==7/30/07==
+I set up digestions for lefty 1106A and righty hemC.  During this two-hour digestion, I miniprepped ABD1-8.  All of the pellets were the darkest brown I've seen yet. One was significantly lighter brown (ABD2) so I discarded this one immediately. I autoclaved two 96-well blocks and added 1mL of LB/Amp to each well. I picked colonies from T7+hemA in BLR and T7+hemAC in BLR. The T7+hemA did not have enough colonies to fill the 96-well block, so I left about three rows empty. I covered the blocks and placed them in the incushaker upstairs and then made gels. I then ran my digests on a gel, gel extracted, cleaned up, ligated, and then heat-killed the ligase for 20min in the PCR machine set at 65C block. I then added BamHI and BglII to eliminate any uncut vector and ran this digest for one hour. Then I transformed into TG1 cells and plated on LB/Amp plates. I also set up test digests with BglII/XhoI on ABD1 and ABD3-5. After one hour, I ran these samples on a gel. However, my bands corresponded to uncut vector 1106A hemA+hemB. I decided to try John's lefty/righty method with this composite part as well so I transformed 1106A hemA+ hemB as well as 1106A hemAC+hemB into the lefty strain and 1106A hemD into the righty strain. [[User:Kdoan|Kdoan]] 17:45, 30 July 2007 (EDT)
+==7/31/07==
+I came in to pick one colony from each of the lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, and righty 1106A hemD plates and grow these in LB/Amp cultures tubes. My 1106A+ hemC had only three colonies! So I picked all three and grew in LB/Amp cultures as well. Austin suggested I look up the toxicity of hemC since I'm having SOO much trouble, but hemC doesn't appear to have any toxicity. I retrieved my T7+hemA in BLR and T7+hemAC in BLR blocks and spun them down. T7+hemA had around five clones that were very dark brown. T7+hemAC had about a third of the clones that were browner than the rest but much less brown than the T7+hemA.  I picked two of the darker ones from each block and grew 2uL of each in LB/Amp cultures. I also made lots of construction files and looked up an old heme article to find out about the LCMS they used to quantify the amount of heme being expressed by their cells. I emailed the article to Mario so he can see if it's doable.  [[User:Kdoan|Kdoan]] 18:03, 31 July 2007 (EDT)
+==8/1/07==
+I came in the night to miniprep lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, righty 1106A hemD, 1106A+hemC1-3, T7+hemA1-2 and T7+hemAC1-2. I came in the following morning and sent 1106A+hemC1-3 for sequencing. Then I digested righty 1106A hemD, lefty 1106A hemA+hemB, lefty 1106A hemAC+hemB, T7+hemA2, T7+hemAC2, and 52+hemB so I can make composite parts. I made gels and after a two-hr digestion, I ran a gel. The 1106A hemA+hemB, 1106A hemAC+hemB, and 52+hemB worked so I gel extracted these, added ADB, and melted the gels. Righty 1106A hemD yielded no bands, while T7+hemA2 and T7+hemAC2 yielded too many. I set up second digestions, but used T7+hemA1 and T7+hemAC1 instead. After two hrs, righty hemD still yielded no bands, while T7+hemA1 got the correct bands and T7+hemAC1 yielded too many bands again. I figured there was no DNA in the miniprep of righty hemD I was working with and grew another clone of righty hemD in LB/Amp culture to miniprep again tomorrow. [[User:Kdoan|Kdoan]] 16:51, 3 August 2007 (EDT)
+==8/2/07==
+I miniprepped righty hemD and ran digestions of righty hemD, T7+hemAC1, and T7+hemAC2, hoping at least one would work. After two hours, righty hemD worked, while the T7+hemAC1 and T7+hemAC2 yielded only one band. I gel extracted righty hemD and cleaned up the digestion along with the digestions that worked from yesterday.  I ligated the respective products together. I transformed T7+hemA + hemB immediately on LB/Amp plates. The lefty and righty ones I heat-killed the ligase at 65C for 20 min, digested with BamHI and Bg1II to get rid of parent vector, and transformed into TG1 on LB/Amp plates. [[User:Kdoan|Kdoan]] 16:51, 3 August 2007 (EDT)
+==8/3/07==
+I chose four colonies from each 1106A hemABD and 1106A hemACBD and grew these in LB/Amp cultures. I then added 2mL of LB media to the T7+hemA + hemB plate and scraped. I then extracted about 1mL and miniprepped. I then performed a plasmid transformation into 90uL of BLR cells and plated on an LB/Amp plate. I helped David autoclave. [[User:Kdoan|Kdoan]] 16:59, 3 August 2007 (EDT)
+==8/4/07==
+My T7+hemA+hemB plate came out very weird. There were lots of colonies but tons of satellite colonies as well. So I took the miniprep of T7+hemA+hemB in TG1 and did another plasmid transformation into BLR cell on LB/Amp plates. Just in case the LB media I used was contaminated, I took the digested/purified products of T7+hemA and hemB and religated and transformed into DH10B cells. I then plated on an LB/Amp plate. I also miniprepped the four 1106A hemABD and four 1106A hemACBD.  I only kept three of the 1106A ABD and two of the 1106A ACBD. I can tell from pellet color that these had not worked.  I decided to test digest with Bg1II/XhoI, but I only ran the digest for 1/2 an hr because I was so hungry. I ran these digests on a gel and got only singly cut vector. I'll have to run these digests again for a longer period of time. [[User:Kdoan|Kdoan]] 00:15, 7 August 2007 (EDT)
+==8/5/07==
+I moved the T7+hemA+hemB plate from the incubator to the fridge. [[User:Kdoan|Kdoan]] 00:15, 7 August 2007 (EDT)
+==8/6/07==
+I autoclaved a 96-well block and added 800uL of LB/Amp media into each well. Then I picked single colonies.  I placed the block into the shaker upstairs. [[User:Kdoan|Kdoan]] 00:15, 7 August 2007 (EDT)
+==8/7/07==
+I retrieved the 96-well block and centrifuged it for ten minutes. When I looked at the pellets, they were less brown than the ones with T7+hemA alone. I knew something had gone wrong so I cleaned up the block and set up new digests for T7+hemA1, T7+hemA2, and 52+hemB to combine them again. I made more gels, then set up test digestions with BglII/XhoI for ABD1, ABD2, ABD3, ACBD1, and ACBD2. After two hours, I ran a gel for the T7+hemA1, T7+hemA2, and 52+hemB. All three got good bands and sizes so I gel extracted T7+hemA1 and 52+hemB, gel purified, ligated, transformed into DH10B cells and plated on LB/Amp. I'm also going to prepare for my assay and do a practice run on ShemA+B and ShemAC+B.  So the first step is to do plasmid transformations of each miniprep into DH10B and plate on LB/Amp as well. I also plated a negative control of DH10B with water and KCM, but no plasmid on a plain LB plate. I then helped Arthur pour and flame plates. [[User:Kdoan|Kdoan]] 15:06, 7 August 2007 (EDT)
+==8/8/07==
+I checked out my four plates and all had ample colonies. I added 2mL of LB media to the (T7+hemA)+hemB in DH10B and scraped.  I then extracted 70uL of the mixture and miniprepped this.  Then I did a plasmid transformation in BLR cells on plated on an LB/Amp plate.  I took 250mL flasks and added 100mL of LB media to one flask labeled DH10B (negative control) and 100mL of LB/Amp media to flasks labeled ShemA+ShemB in DH10B and ShemAC+ShemB in DH10B.  I then picked one colony from each corresponding plate and grew in the correct flask. I then placed these flasks in the incushaker. For the rest of the day, I updated the wiki and registry with many many parts. [[User:Kdoan|Kdoan]] 13:39, 8 August 2007 (EDT)
+==8/9/07==
+I looked at my three 250mL flasks and noticed the DH10B negative control flask was clear. Maybe I didn't pick the colony or accidentally grew in LB/Amp media. So I plated more DH10B competent cells on an LB plate. The other two 250mL flasks containing ShemA+ShemB and ShemAC+ShemB were brownish and cloudy so I placed these flasks in the fridge. I looked at my T7+hemA+hemB in BLR and noticed only six colonies. So I performed a second plasmid transformation of my T7+hemA+hemB in DH10B miniprep into BLR cells and plated onto an LB/Amp plate. I also made more gels. [[User:Kdoan|Kdoan]] 13:54, 13 August 2007 (EDT)
+==8/9/07==
+Nhu took my DH10B negative control plate and T7+hemA+hemB in BLR plate and put them in the fridge. Thanks Nhu! [[User:Kdoan|Kdoan]] 13:54, 13 August 2007 (EDT)
+==8/12/07==
+I picked a colony from my second DH10B plate and grew it in 100mL of LB media in a 250mL flask.  I placed the flask in the incushaker. I looked at my T7+hemA+hemB in BLR plate and this time, there were many colonies. I obtained two 96-well blocks from upstairs and added 1mL of LB/Amp media to each well. I picked colonies from the T7+hemA+hemB plate in BLR and grew one in each well. I tried to avoid the colonies that were white and picked the ones that were more beige-colored. I placed the gauze sticky over both and brought them upstairs to the special incushaker. [[User:Kdoan|Kdoan]] 13:54, 13 August 2007 (EDT)
+==8/13/07==
+I obtained my two 96-well blocks from upstairs and spun them down in the large centrifuge.  I picked two from each block that belonged to the group of wells that were browner and darker than the rest. I grew these in LB/Amp culture and placed the four tubes in the incushaker. I kept my 96-well blocks in the fridge just in case. My DH10B negative control flask was cloudy this time so I gathered the three 250mL flasks and performed a UV-Vis to measure the absorbances. I spun down 5mL of each culture, flicked out the supernatant, resuspended in 500uL of EB buffer, added 300uL of P2 lysis buffer, 300uL of 0.5M HCl, centrifuged at 3750rpm for 10min, then extracted 1mL from each supernatant and transfered to labeled plastic cuvettes. I also had a blank with EB buffer, P2, and HCl. I took these cuvettes to the Beckman machine upstairs, set the wavelength range to 250-800nm, and measured the absorbance for the blank first.  Then, I measured the DH10B, ShemA+ShemB, and ShemAC+ShemB.  The computer plotted the lines on one graph and I printed out the data. I then completed updating the registry and my notebook.
+[[User:Kdoan|Kdoan]] 14:02, 13 August 2007 (EDT)
+==8/14/07==
+I miniprepped T7AB1-4 and ran test digestions with Bg1II/XhoI. After an hour, I ran a gel with these samples and all four got the correct sized bands! I planned out my digestion for tomorrow and asked around about the LCMS. I also prepared a little for the presentation on Thurs. [[User:Kdoan|Kdoan]] 14:50, 14 August 2007 (EDT)
+==8/15/07==
+I digested T7AB1-3 and 51+hemC with the appropriate enzymes to combine them. I did multiple T7AB and two 51+hemC digestions to ensure at least one works. I made more gels and prepared for the presentation. I also helped David with autoclaving. After two hours, I ran the digestions on a gel. I got the right sized bands for all except T7AB2, but T7AB1 and T7AB3 were good. I gel extracted and combined the two 51+hemC into one tube.  I gel purified, ligated T7AB1 and T7AB3 with 51+hemC, transformed into DH10B, and plated on LB/Amp plates. [[User:Kdoan|Kdoan]] 17:15, 15 August 2007 (EDT)
+==8/16/07==
+==8/17/07==

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Kristin Doan Notebook

From 2007.igem.org

Latest revision as of 21:15, 15 August 2007

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