2 replies to this topic

[sara.r]

Hi
when adding restriction sites to a primer is the flanking sequence at the 5 end of restriction sites important and how many bases are required? Actually I have heard that new england biolabs suggests best sequence for each enzyme but I couldnt find it.

best

[phage434]

Check these two NEB pages:
http://www.neb.com/n...nucleotides.asp
http://www.neb.com/n...ized_vector.asp

I usually add 8 bp, but 6 is probably more than enough.

[scolix]

you donot need a specific sequence but a number of bases on either side of the restriction sites. I use 6 extra bases, but for some enzymes you might need 8.
NEB has the number of bases required for each enzyme.