Restriction Digest Protocol

2015-01-13

azim58 - Restriction Digest Protocol

Basic outline for a restriction digest: may want about 20 uL volume with
500 ng plasmid, BSA, Buffer, water, 1 uL enzyme; if performing a
sequential digest move from lower to higher salt;

Note that if a vector has been digested, and a ligation will be
performed, then the alkaline phosphatase reaction to prevent self
ligation can be performed right away in the same tube. Then all of the
enzymes can be removed, and the correctly sized fragment can be purified
in a gel purification reaction. see Promega CIAP Protocol. Also note that
in this situation it may be a good idea to let the cut vector run on the
gel for a long time to make sure that all of the cut vector is well
separated from the uncut vector.

Note that when doing a sandwich gel extraction after a restriction digest
it may be a good idea to run two totally separate identical gels so that
it is as easy as possible to align everything.

Resources

* fermentas buffer conversion chart.pdf
"C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2014\01-19-2014d1132\01-19-2014d1201\fermentas buffer conversion chart.pdf"

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