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.
That depends upon the enzyme. you can check a table in NEB catalog.
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).
Could you post the others Phage? Do you have a source of this information or is it just experience?
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.
NotI is the other common one. See here:
Hmm, interesting. Never seen any more than 6 extra bases before. I do clone with NdeI and NotI often enough though with 6 flanking bases and don't recall many problems. Thanks for the info, i'll keep it in mind.