Template:Team:UC Berkeley/Notebook/DV notes

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Revision as of 02:17, 21 June 2008 by Dirkvandepol (Talk | contribs)


Dirkvandepol 17:08, 12 June 2008 (UTC)

Today I will explain what I have been doing for two weeks. It will not be impressive. Yesterday I completed ---I was about to say I finished fixing my mistakes, but truthfully what I did was properly document the fixes Jin made to my mistakes. I completed that work and sent it to Jin at 11:56 PM. Then I rode my bicycle to my car, loaded my bike onto my car, and drove home.

Dirkvandepol 21:19, 17 June 2008 (UTC)

Yesterday- I helped our two new high school students, Sherene Cheung and Cici Chen, get used to what would be done in the lab. We watched videos of Dr. Anderson's lectures from the previous week to the rest of the students in the lab. Then we ran out my PCR products from the previous Thursday on one of those e-Gels. Most of them worked, suggesting that the primers were good. However, I may have made a couple of mistakes in the primer setups that I made-- I may have PCR'ed up the sequence I was to mix, and I may have failed to PCR up my <b1006> sample. I will do that with the next time I run PCR, which will be quite soon, because I forgot to run a PCR using a pair of Aron's Oligos that were emailed to me. After doing a gel purification using the gel, my minions Sherene and Cici set up a restriction digest. When that was done, I purified the digests using a Zymo-column off the shelf purification system.

Today- So far, we have run a set of PCR's and run a mix using oligos dv009 and dv010. The PCR's were the three that were included to make the <Phns> promoter. Since the dv003 oligo used the first time still contained a restriction site, we used the replacement dv003 to remove that site. I chose to re-do all three of the PCR's involved because I had a labelling mixup that I wanted to clarify- while doing the zymo column purifications yesterday, I got a little mixed up with labelling- I thought I had labeled something that I actually had not, so I got confused about <Phns> fragments 2 and 3. I had originally planned to ligate 1 and 2 together, but my solution to my mixup was to instead to ligate 2 and 3 together, but, since 3 had to be replaced, I would therefore have to re-do the other, because I wasn't sure which vial was 2 and which was 3. So I just had my minions re-do all of them.

 Also, we bound oligos dv009 and dv010 (forward and reverse of the His-tag> part) together by mixing- we used a special protocol on the PCR machine which we entitled "Mix":
    Heat to 94C for 2 min.
    Cool to 37C forever,  using a ramp-down rate of -1C per second
 When this was done, we did an Eco/Bam restriction digest of our mix.
  For the rest of the day: 
   -Zymo cleanup of the His-tag> product digestion
   -Ligation of yesterday's cleanup products
   -PCR up the Aron Lau oligos using pBca1037 template
   -PCR up dv021 and dv022 
   -Transform ligations
   -maybe some other stuff, stand by...

Dirkvandepol 02:20, 18 June 2008 (UTC)

  Before we left, we PCR'ed together the product of our Phns PCR's, fragments 2 and three were PCR'ed together by SOEing PCR.  We also did the Zymo cleanup, the ligations, and the transformations.  I still haven't PCR'ed up the Aron Lau Fragments and the dv021/022 thing to make <b1006>.  I'll have to do that tomorrow.  Maybe I can get here early.


Dirkvandepol 18:30, 19 June 2008 (UTC)

(that's 11:20am PST, june 19...) So the Tues, Jun 18 SOEing PCR of Phns fragment A and E had initially failed (The Aron Lau- primer reaction to amplify SpvR succeeded, though); I set up eight attempts at a repeat of this reaction, using DMSO, 45 degree annealing temp, an alternative tube of fragment A (labelled "Phns 1"), and all combinatorial permutations thereof, for a total of eight reactions. All were success, but doing the work, and consulting with Jin has raised a specter of doubt on whether I used the right primer to make fragment E. We will digest two of our tubes (2 and 4) and then, rather than do a zymo cleanup, we will do a gel puficiation to confirm that I didn't use the bad primer to make fragment E (which, if I had, would show two bands when run on the gel for it having been cut by EcoRI). If a single band is obtained in these lanes, then we will ligate and transform, hopefully today.

 While the digestion is digesting, we will watch day 2 of the videos, hopefully somewhere downstairs.

Dirkvandepol 02:17, 21 June 2008 (UTC)

715pm PST Yesterday, those gels showed a single band, which we sampled. In the evening, after Cici and Sherine left I zymo-cleaned, ligated, and transformed SpvR and Phns. The plates today showed colonies, which have been put into a culture block. Tomorrow Mahdvi will mini-prep them. I don't expect I'll come in. But maybe I will.