General question about restriction sequences in PCR primers - (Feb/12/2008 )
I plan to put restriction enzyme cloning sequences in my primers (not a very hard task )...and as it is well known, I also have to put additional sequences to increase the cutting efficiency at the primer termini. I’ve noticed that the stratagene (http://tinyurl.com/2c885o) and NEB (http://tinyurl.com/2yvp6r) catalogs use the same additional bases for a given enzyme (e.g. for EcoRI : CCGgaattcCGG). So I’m wondering if the nature of these bases is important by itself, or it is only the number of bases used…Concretely, can I use AAAgaattcTTT, ACCgaattcGGT or whatever (but still keeping in mind the classic encountered problems in primer design, e.g. hairpin, self-dimerization, etc.) ? It should help me to design suitable primers ()
You might want to avoid long stretches of A/T, which will have low Tm and tend to breathe during PCR reactions, but otherwise, I believe it is a don't care. We use the 8-base sequence GTTTCTTC at the 5' end of primers. This sequence was shown to be optimal for A tailing of Taq-based PCR products. The thought is that you can TA clone the PCR product easily if necessary, rather than directly cutting and ligating into a vector.
we have always used 6 A's or T's at the ends of primers for proper digestion. it has always worked for us. People use different sequences at the ends but we haven't tried other sequences.
Thanks for your answers !