How many bases should be added to end of enzyme in PCR for effective cutting - (Jun/02/2008 )

I am cloning a Bacmid and have made primers with and addition of a specific restriction enzyme which corresponds to the insert I want to use later. My forward primer has 3 bases before the enzyme and the reverse primer has 6 bases before the enzyme therefore these will be extra bases outside of the cut site once I do a digest of the PCR product. Does anyone know how many bases I should have on the ends to get a good cut. I have tried to digest my PCR product and self ligate but have had no luck. Any suggestions would be appreciated.

I am cloning a Bacmid and have made primers with and addition of a specific restriction enzyme which corresponds to the insert I want to use later. My forward primer has 3 bases before the enzyme and the reverse primer has 6 bases before the enzyme therefore these will be extra bases outside of the cut site once I do a digest of the PCR product. Does anyone know how many bases I should have on the ends to get a good cut. I have tried to digest my PCR product and self ligate but have had no luck. Any suggestions would be appreciated.

Try here

6 extra bases is the general rule - that should cover any digest. Excessive maybe but for the price of a couple of extra bases, save the hassle.

Except for a few, like NdeI, that need more than six. I use 10. As you say, they are cheap. Don't forget that if you have a double digestion, this is also the minimum distance between the two cut sites (in a multiple cloning site, for example).

Thank you everyone for your great suggestions. The enzyme I am using is BSU36l and I understand it does not ligate well at the best of times. I looked on the NEB web site but did not find it listed there for number of extra base pairs. I will definitely try the suggestions made for using 6-10 extra bases. Thanks a lot.

Alabgeek

NotI is the other common one. See here:
http://www.neb.com/nebecomm/tech_reference...nucleotides.asp