User:Anthony Salvagno/Notebook/Research/2011/02/14/Finishing Unzipping Construct:Ligation Attempts

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Results from Last Week

Well I had a misstep in the lab. While I was purifying the Adapter-DpBR ligation (aka ApBR) I felt a splash as if I got some of it on my face. Now after purification I did get some result so the only way to know what I got is to do the final ligation and gel analysis. While this is going I will run the other ligation simultaneously.

Anyways the nanodrop says I have 40nM of ApBR. We'll see.

Unzipping Ligation

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdExRV3M2RzJyUmktc2djcndTSEtkOGc |width=500 |height=300 }}

Adapter-DpBR Ligation 2

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdGJoTi1GVGVBWXpvWVpmMFlFQjRFbkE |width=500 |height=300 }} Same ligation as Friday, and I'm using the same recipe so I don't feel the need to do anything differently notebook wise.

Results

I also did the pALS digestion with BstXI so we'll have to see how that goes, but the cleanup of both reactions (digestion and ligation) are done so here are the concentrations for tomorrow's reaction:

  • DpALS - 331ng/ul --> ~115nM
  • ApBR - 158ng/ul --> ~55nM

Better than Friday's results.

Also the results of the unzipping ligation are in as well. I got 3kb band with no product (meaning there is no bstxi site in the 3kb biproduct). I also got a skewed 4.4kb band. This is because pBR322 and pALS are about the same length but pALS is a little longer so the two bands were beginning to separate but not by much. Finally I got a 9kb band which is what I should get. It was probably about half as bright as the plasmid bands suggesting incomplete digestion/ligation. Will do another run tomorrow.

Steve Koch 00:56, 15 February 2011 (EST):This could be really good, right?
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