Can i double digest with HindIII and BamHI enzymes that have just 10 nucleotides cut site distance in MCS of vector for insertion? How about sacI and EcoRI with 12 nucleotides cut site distance?
Thanks for the help.
Yes. Both enzymes will cleave MCS.
Usually, restriction enzymes used in cloning dissociate from DNA and thus do not interfere with action of another enzyme. The only issue here might be that some restriction enzymes are not efficient if their site is close to the DNA end. This varies from enzyme to enzyme, but leaving ~6nt from the end is safe in most of the cases. More information can be found on the supplier's web site:
https://international.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
But you will not be able to distinguish DNA cut by both enzymes from that cut only by one on a gel.
Thank you for your help. Those RE sites are not close to the DNA end. The only issue that i am worried about is that i can not do the double digest with those RE.@
Actually, if you use New England Biolabs HindIII-HF and BamHI-HF, they will cut 100% together in CutSmart buffer.
Thanks you dear Gallery.
I'm just worried about distance between two REs, to do a double digest. I have been thinking that REs needs some space to restrict efficiently, so what is the minimum distance between two REs site?
Is there any documents about The minimum distance (bps) needs for each RE?
I'm not talking about REs that are close to end of DNA.
May i fail in double digest reaction just because of REs have not the minimum distance that they needs?
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