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

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Digestion

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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.

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.