Retroviral Vector won't digest ideas! - (Oct/22/2005 )
I have been trying to drop a 3800 bp fragment out of a 1100 bp retroviral expression vector using a standard double digestion protocol. The RE's in question are Not1 and Xho1 which supposedly are compatible. The digest went overnight at 37 degrees but when the samples were run on .7% agarose...the Not1 didn't seem to do anything (single digest), Xho1 seemed to digest but not always to 100% (single digest), and no band dropped out when the two were mixed.
Anyone have any suggestions or tricks? Is it possible that the large amounts of vector are supercoiled and I need to add more enzyme?
Very far-fetched, but could this plasmid be from a source with CpG methylation activity (of course bacteria usually don't CpG methylate).
Other perhaps more likely explanations:
-impurities in plasmid preparation
-enzymes too concentrated (glycerol from commercial preparations of endonucleases may inhibit reactions).
-enzymes have been inactivated (e.g. someone else left it at room temperature for some time, or enzymes merely too old).
-buffer not well put back in solution before use (some crystal left undissolved).
-error in sequence of plasmid