Designing a Primer with a RS that has a W - (Jan/11/2011 )
So i am in the process of trying to amplify a promoter and also adding restriction sites HindIII on the 5' end and SexA1 on the 3' end. Can someone tell me if this looks okay:
Forward: AAAAAA AAGCTT
Reverse: AAAAAA ACCWGGT3
(W = A or T)
How does it look?
Couple things you might want to consider. First, according to NEB HindIII prefers pyrimidines as the overhang for digestion close to the end of fragments, so you might want to consider 3-5 C or G bases rather than the A. They don't specify a preference for SexA1. Also, on your reverse you have written the complimentary 3'->5' sequence and this actually needs to be the reverse compliment sequence in the 5'->3' direction. You really need to make sure you have the correct reverse compliment sequence in order to get the product you are needed. If your promoter is only ~20 bases, why don't you post the entire sequence and primers you are designing. Plus, you will be much better off just ordering purified oligos and annealing them. This way you can order them with the restriction site overhang (as if you digested), anneal and ligate into your digested destination vector. This will make things much easier and faster!!