Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

restriction enzyme flanking sequence preferance - (Feb/20/2010 )

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

-sara.r-

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

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

-phage434-

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.

-scolix-