Has anyone come across a situation when you get a amplified band in PCR, using a recombinant vector as template (tested alongside the positive control; genomic DNA as template) but no release of fragment when digested with appropriate restriction enzymes. Any suggestions please?
Dear Satya,
Have you thought about DNA-metylation sensitve restriction enzymes? Which Enzymes are you try in to use and from which bacterial strain were they isolated?
best Soenke
How big is your band, and your target? Did you have controls for your enzyme digest? If it's a double digest, you will need a separate control for each enzyme. If you run controls and they are cut, maybe the restriction site is not present in your template.
Following above question... I am using pET28a(+) vector ligated with a gene of Mycobacterium smegmatis. I have used routine HindIII/NdeI (NEB) combination with 2.1 buffer and also used control. The combination has worked for another recombinant vector (pET28a(+) with other mycobacterial gene) with approx. same size (~1kb) of insert. I didn't thought about DNA-metylation of restriction enzymes, may be that trick works, need to check. Thanks for the suggestions.
Scince HindIII and NdeI are not methylation sensitve. I do not think that That is the reason. At least an opening of the vector should occour. I think you should either go back into the sequenze and cloning process to rethink the whole cloning, and/or you should sequenz the clone you are working with and than you will see why the restriction does not happened.
Kind regards Soenke
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