User:Anthony Salvagno/Notebook/Research/2009/09/16/Ligation of Construct to pBR322 Long Strand (If this doesn't work I'm going to fucking kill someone)

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I like these long titles...

Setup

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Run at 16C. Every 30min I will add 5nM (which turns out to be about 2.5ul) to the reaction. This will prevent bullshit from going on. I will do this until the reaction has 20nM (maybe I should go to 25nM?) which will be at the 1.5 hour mark and let it run for another 30 min.

I also changed the amount of T4 Ligase to 1.5ul instead of 1ul.

  • Just added the 1st extra 5nM (2.5ul)
  • Just added 2nd 5nM T+B - should now be at 15nM
  • Added final 5nM T+B - should be up to 20nM or something like that.

Gel

I have 57.5ul of reaction plus 15ul of 5x loading dye to give me 72.5ul total. I will probably have to put this in 3 lanes or so. Now I know why Koch taped the lanes together and is something I should have done. Lesson learned.

I put the DNA in 2 lanes. I should have done 3 but I wanted to push it. Stupid me. Oh well, hopefully I don't lose too much. Maybe on the order of like 2-5ul which isn't bad for a 73ul reaction.

I'm running the gel at 130V but after about 10 min put it up to 135V. One day I'm going to see how high I can crank it before it melts. Hopefully today is not that day. I took this image after about 45 min of gelling. I'm letting the gel run a little bit to see if the bands separate some, but it doesn't look promising. Now what?

OWW doesn't seem to be uploading pictures at the moment, but the second picture looks worse and the camera magically is darker. Fuck Life!

Other Notes

As a precaution I am digesting 10 more ug of pBR322 for use tomorrow. Thanks Brigette.

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