Primer design: how do I add a restriction site to my primer? - (Mar/17/2008 )
I know how to design primers and how to add a restriction site. However I am looking for a good source that tells me how many extra nucletides I need to add at the end of the primer so the enzyme can cut. This varies from enzyme to enzyme and I have short list but I am looking for a list of as many enzymes as possible. Any ideas where I can find this information?. Thank
I have these files from New England Biolabs.files are attached
In general I would add between 6 and 9 nts
I usually add 4 nucleotides
5' GGCG-GAATTC-CCACC-ATGCTGCTGGCAGCGAG -3'
overhang- RE - Kozak seq- Gene specific sequence
If my restriction enzyme(-s) still have problems cutting, I do an intermediate blunt end ligation of my PCR product into a vector (using f.ex. Blunt TOPO-kit). And then I can cut the PCR product from this vector using my RE of choice.