primer design with restriction enzyme - (Jul/22/2014 )
I'm designing my primers to clone the entire coding region of my gene of interest (GOI). I understand we have to add flanking sequence at the 5 end of restriction sites. Does anyone kow how many bases to add for the enzyme EcorV? Also, can we add any random bases or do they have to be of particular sequence. The NEB website only sussgest the flanking sequence for some enzymes and not all.
I'm uisng primers BamHI and EcoRVI. The NEB web site suggest the flanking sequence for BamHI and not EcoRV.
For example for BamHI, NEB website suggests the following bases to flank the BamHI restriction site:
(BamHI, ggatcc): ccg gga ttc cgg
Forward primer using BamHI:
ccg gga ttc cgg GCCGCCACC (kozac sequence) ATG(start codon)+ 20 bases of GOI
Reverse primer using EcorV:
gaat atc TGA (stop codon) + 20 bases of GOI
Also, I've got to add the stop codon in the reverse primer if I want to clone the entire coding region without tags?
In general, the specific bases are unimportant, but the number of them can matter. I would always use six or more, just because they are inexpensive, and then I don't have to think much about it. I don't know of any enzymes that require more.