So, i am double digesting pRSET A vector and an insert with BamHI and PstI ... running a gel and purifying the respective bands.... After ligation and transformation , and running PCR on the colonies with the vector's T7 primers I'm getting a band with the size of what would be the amplicon if the vector self ligated and took no insert
AND IT'S DRIVING ME MAD
Because how can a vector cut with 2 different incompatible REs and gel purified (so the section removed would migrate in the gel being smaller and id supposedly have a cut vector free of the cut section or am i wrong on this?) self ligate?
I just read somewhere that some of the vectors might be cut with only one of the enzymes and those are the ones self ligating.... Is that the case?
It could be, as mentioned above, that you had some DNA that was only cut by 1 enzyme. It also could be that in your plasmid preparation was a tiny fraction of some plasmids carrying a deletion of one or both sites. Remember that a contamination level of either of these at only a fraction of a percent would still give you lots of transformants.
I have a few suggestions for you:
1. Run 2 controls where you digest your vector with only one enzyme. Are both enzymes active and able to linearize your DNA?
2. If the answer to #1 is yes, then repeat your digest. After the digest, treat the plasmid with a phosphatase enzyme (either CIP or antarctic phosphatase). This cleaves off the phosphate that is necessary for self-ligation. However, the insert still has its phosphates (assuming you don't treat it with the phosphatase enzyme as well) and can be ligated into the vector.
Thank you james, yes both my enzymes are definitely active since i cut the insert from the donor plasmid with them too
Im either going to use a phosphatase like you suggested or cut the vector in the MCS with another Restriction enzyme after ligation ... whichever i have lying around in the lab :)
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