How many bases does it occupy in the DNA in search for the recognition sequence?
I am trying to digest with BamHi and XbaI using a 73 bp fragment and am not getting the clones I need. A: 1 base for BamHI, 2 bases for XbaI, and for other enzymes it can vary between 1-5 bases. This answer took some searching but I finally found it in a great experiment performed by Fermentas scientists described here. To summarize, PCR primers were designed with 1-5 bases added on next to enzymatic recognition sequences and were P32 labeled and used to amplify fragments using PCR. Restriction digests were performed, products separated by 10% PAGE and the % efficiency of cutting was determined. You will be happy to know that BamH1 had 50-100% cutting efficiency with just 1 base next to the cleavage site and XbaI needed 2 bases flanking the cleavage site to achieve 50-100% cutting efficiency. So if your fragment has >2 bp flanking the cut sites, the enzymes should be cutting correctly. To be safe, you can always extend the ends by 5-10 bp and you should be totally safe. Q: Does phenol
