Question about NcoI digestion... - (Aug/15/2005 )

I'm currently trying to digest an 1800 bp PCR product with NcoI and BstEII, and am having trouble ligating the purified fragments. I was wondering if the NcoI site might not have enough flanking bases for the enzyme to properly digest it. Currently, the forward primer I am using is 2 bases on the 5' end longer than cut site ( 5'-GA*cutsite*rest of primer*-3' ). Is this sufficient for NcoI, or should I use a longer set to ensure proper cutting?

Hi,

NEB has an excellent resource for questions like this here:
http://www.neb.com/nebecomm/tech_reference...ed_vector.asp#N

It's a little misleading, however. Although it's recommended to have 2 bp upstream of your restriction site on your PCR primers, the fine print above the table mentions adding 4 extra to any of the recommended numbers when designing PCR primers.

Hope this helps,
Hank

Yes, I had reference that table in the catalog, but was confused on why there was that 4 base discrepancy between the two. I had thought that a fragment like mine would be effective with 2 past, but I may just try again with 6. Thank you so much for the quick reply.

It really is confusing and it's something I think NEB has to put more emphasis on highlighting in their catalog/webpage.

In general though, I always add 6 bases upstream of my restriction site when designing primers. Better to have extra bases than not enough.