Kristin Doan Notebook
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| + | So I realized that I've been adding 1ul of PCR buffer instead of 5ul. This is not good cause 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 D | ||
Revision as of 21:49, 12 June 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. 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)
6/7/07
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
6/8/07
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)
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 cause 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 D
