User:Anthony Salvagno/Notebook/Research/2009/04/01/Digestion and Double Digestion

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Digestion

Gel Prep
Tube Number Vol gDNA #/Vol Buffer 10x Vol BSA 10x Vol XhoI EcoRI Vol H2O Total Vol
1 32uL 2/10uL 10uL 5uL -- 43uL 100uL
2 32uL EcoRI Buffer/10uL 10uL 2.5uL 2.5uL 43uL 100uL

After 2 hours I will add extra enzyme. 4uL XhoI to tube 1 and 2uL of each enzyme to tube 2.

Reasoning

We are going to digest with XhoI as normal. Hopefully using a smaller vector like pBluescript will allow for successful ligations. Apparently E coli doesn't like large sequences, so that explains why the previous attempts didn't pan out.

The double digest has a different use, but still ultimately the same. I think we want to clone using pRS413 still (for this case) and cutting with 2 enzymes should give us smaller fragments. Comparison on a gel between the two digests should determine if the fragment sizes get smaller. Steve Koch 17:42, 1 April 2009 (EDT): I still don't think it's worth the effort for the double digest. I don't expect we'll ever want to chromatinize these plasmids. Plus, 50%-ish of your double-digested genomic DNA will be unclonable (it will have Eco-Eco or Xho-Xho ends.

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