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

{{#widget:Google Spreadsheet |key=0Agbdciapt4QZdFVSaFRka0VaUWhFZ3NTWlZ2MV9Jc1E |width=500 |height=200 }} {{#widget:Google Spreadsheet |key=0Agbdciapt4QZdE5hb1IwVFNRZVE4X2dtSFE0Vnp2TXc |width=500 |height=300 }} 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. {{#widget:Google Spreadsheet |key=0Agbdciapt4QZdGJoTi1GVGVBWXpvWVpmMFlFQjRFbkE |width=500 |height=300 }} I will get to the other ligation, the final ligation on Monday. It all goes according to plan... mwah ahahahahaha!!!