User:Anthony Salvagno/Notebook/Research/2009/06/17/Unzipping Planning

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Meetings with Koch always revitalize the mind. I really enjoy talking about everything the lab is doing, even things that I am not part of (even though everyone has some aspect of each project to work on). Anyways we talked about SDM, and I am back on the case after a little break of not doing much.

Previous Work

Even though this entire notebook is of previous stuff, I need to gather my thoughts to be able to replicate that stuff in a different lab.

  • Create random yeast fragments
  • Insert fragments into plasmids (pbluescript in my case)
  • Clone fragments
  • cut plasmid-frags with RE
  • ligate pfs with oligos (3 different)
    • top and bottom oligos get annealed together
    • hairpin oligo needs to self anneal
  • gel extract ligation products


I have tethered before, but I have not yet tethered my ligation products. For this you:

  • flow anti-dig
  • flow BGB
  • flow DNA
  • flow microspheres

To Tether Right Now

If I wanted to tether right now, I would need the following things:

  1. tetherable DNA
  2. good microspheres
    • by this I mean they would have to be streptavidin coated and not clumpy.
  3. anti-dig
    • I don't know if this stuff goes bad
  4. BGB
  5. popping buffer

We have:

  • all of it except:
    1. popping buffer
    2. BGB

The popping buffer is at Osley Lab. I'd have to learn to make BGB, but apparently all the stuff for that is here. Theoretically tethering is possible, but publishing capabilities is probably at 35% for a low estimate. I need to do a few test runs to make sure everything is ok.

If I Had to Optimize...

I would need to order the oligos used above and get the DNA glycerol stocks from Osley Lab. The reason for this is this. Look at that shit! It's awful. There are bands all over the place. I will need to make it look better. There is a chance one of the tubes I have is nice solid tetherable nonconcatenations of DNA, but I don't know. I will have to look in my box to see how I gel extracted.