Hello everyone; I have a issue that is seeming to be very challenging: I have to cleave an insert from a recombinant plasmid into pQE60. I have done some analysis on Webcutter and discovered that the option that I have is to use Nco I and Bam H I. But the sites are so closed to each other in the vector. I do I achieve my aim: I am using NEB HF enzymes
According to the NEB website BamHI-HF will cut 50-100% with 3bp past the cut site and NcoI is the same. So these two enzymes should be compatible as long as there is some extra overhang 3+bp after you cut.
I am a bit unclear of your goal. I assume you are trying to open the vector to insert a piece of DNA between these two sites.
You can try doing a sequential digestion with BamHI first and then with NCO I. It should work. Good luck
or try using a plasmid with an insert in that plasmid already - this might work http://www.addgene.org/12553/
As a general rule , 6 base pairs should be added on on either side of the recognition site to cleave efficiently. The extra bases should be chosen so that palindromes and primer dimers are not formed. In most cases there is no requirement for specific bases.
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