Kristin Doan Notebook


Revision as of 21:06, 28 June 2007 by Ayu (Talk | contribs)

My Construction Files
My Sequencing Files


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. who knows? so instead, we worked on our wikis (obviously) The day wasn't in total vain because I did read two articles on heme so I understand better now. yippee! Kdoan 17:37, 6 June 2007 (EDT)


This day was super hectic. We all 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


2nd day: things were in a little more control today. I was scared to look at my transformed plates. BUT hey, there were colonies! for both plates! and they were white! Very small ones, so 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 I need to get rid of. 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. Once I added all the ingredients to the master I divided each master into four tubes. 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 made sure that each colony I extracted from corresponded with the right number. 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)


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)


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)


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)


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.


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.


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.


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. 17:42, 18 June 2007 (EDT)


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.


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.


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.


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.


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.


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.


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.


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.