User:Anthony Salvagno/Notebook/Research/2011/02/11/PCR Results, Digestion, and Ligation

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PCR Results

I don't have a notebook page for this, but I ran a PCR reaction based on results I got from a PCR reaction I ran 3 days ago. The first PCR reaction gave me more of my product and that other 3kb product and produced no other bands. The reaction I ran yesterday had less 3kb than in the previous experiment and way more 4.4kb product which is what I want. I ordered some perfect match yesterday to help out the reaction and I will run another reaction once that gets here.

Digestion

View/Edit Spreadsheet
View/Edit Spreadsheet

pBR digestion successful. There was only one band of product. Moving on to ligation.

Nanodrop Results

I'm calling this plasmid pALS from now on per Koch's suggestion. It has a nice ring to it. Also to keep track of what I've done to it I will add a letter in front of the pALS to label what stage it is. TpALS is tetherable PCR product with dig-bio for stretching. DpALS is digested with BstXI and is converted to a dig anchor. UpALS will be unzippable pals if I ever call it that (perhaps this will be where I ligate the anchor back onto itself).

pALS PCR:

  • from yesterday: 245.1ng/ul --> 11.764ug DNA in 48uL
  • from two days ago: 156.1ng/ul --> 7.492ug DNA in 48uL

I used 30ul from that tube and digested it with BstXI as per above and my new yield is:

  • DpALS: 64.2ng/ul --> 3.082ug in 48ul
    • this means I lost about 1.5ug of DNA. ~63% efficiency, not too bad.
  • DpBR: 282.2ng/ul --> 98.4nM

Ligation

I am going to do this as a two piece ligation and I'm going to start with ligating the pBR322 digestion (which I still have to do) onto the adapter duplex. Clean that reaction and then ligate DpALS to DpBR. Reactions to come.

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I will get to the other ligation, the final ligation on Monday. It all goes according to plan... mwah ahahahahaha!!!

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