- I have cloned a 1 kb gene into a 5.3 kb vector (in between BamH1 & Xho I) and screening of the recombinant plasmid has been done by colony PCR using gene (insert) specific primer where I am getting the expected size amplicons (1 kb). After that, plasmid DNA was isolated using a Miniprep kit. Again, the extracted plasmid was subjected to PCR using gene (insert) specific primer and got the expected size amplicon. However, when I have done the double restriction digestion with BamH1 and Xho I, there is release of the insert (1kb) from the vector (5.3kb) but along with that, I am getting an extra sharp band of around 3.5 kb. Kindly give me some suggestions.
Dear Deep Prakash Saikia
Are you sure if you have loaded appropriate concentrations of the restriction enzymes in the restriction digestion mixture? It seems to me that you are obtaining uncut supercoiled plasmid band in your restriction digestion product. The supercoiled plasmid DNA migrates faster than the linearized plasmid DNA and this band is the most intense if you run an uncut plasmid DNA.
Your problem may arise due to:
- high amount of plasmid DNA, so the DNA left uncut
- low concentration of restriction enzyme
- too old restriction enzymes, reduced endonuclease activity
Please check the concentration of your plasmid vector, and prepare a restriction mixture according to the manufacturer's protocol.
Best wishes
Getting a pcr product only proves that some of the plasmids have your insert so it is possible that some plasmids exist without the insert and you were just detecting the positive clones, Running a gel with uncut, cut with one enzyme, cut with second enzyme and cut with both enzymes might give you an idea where the unexpected band is coming from but I think that Satyendra Mondal is probably right that it is one of the faster moving isoforms of the empty plasmid
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