Double Digestion with Single Restriction Enzyme - (Jun/15/2007 )

Is there a sppecific protocol that I can use to cut my plasmid with my insert that has the same RE sites on both ends?
I am also having trouble re-isolating the plasmid with insert from transformed cells that were frozen , they previously showed to have the insert that was verified using PCR with gene specific primers. Does anyone have any suggestions as to what I could do? I am kinda stuck with this problem.
Thanks...

If your insert is in a vector with identical restriction sites at each end, you can cut it just as you would normally. You just need that enzyme (it will cut at both sites -- and anywhere its recognition site is present), buffer, and your sample. Depending on which enzyme it is, you may also need BSA. You can find protocols at the New England Biolabs website.

In terms of isolating plasmid from a frozen strain -- are you using a kit? That is the easiest, although there are lengthier protocols. If you are, and still are having trouble, does your plasmid have an antibiotic resistance gene on it? If it does, it is critical that you include this antibiotic in the media in which you grow your bacteria, otherwise the bacteria will "spit out" the plasmid. You need to add the antibiotic to provide "selective pressure," or a reason for the bacteria to keep it (it needs the resistance gene).


Amanda

there is no specific protocol for this. you should know the sequence of your insert and find out a suitable restriction enzyme that will cut you insert in both ends. and you can search for the specific enzyme in vector MCS too