pro-PCR with only one primer... - to check directionality. (Jun/26/2006 )
i did single HINdIII ligation....before checking by digestion i want to check by colony PCR.....i have to use one primer right? forward primer..... please confirm this to me....so that wrong direction insert will not be multiplied ...am I confusing something?
please anyone!!! im beginning to think that you cant actually PCR with only one primer... ...i have done 16 samples and use too much Taq....still have no results but pretty sure that its not going to work... i feel so bad....
You need one primer on the vector, and another on the insert. When they face one another, amplification will occur, when they face the same direction, nothing happens.
A single primer (unless it binds to two locations) will not amplify exponentially (it will create single stranded copies linearly, aka asymmetric PCR).
thank you very much i misunderstood this point....i see it clearly now....however our lab doesnt have vector primers...usually you get primers with the vector?
Almost all vectors have M13 or T7 primers of some variety defined. Your lab likely has the M13 forward and backward primers sitting around somewhere, or they can be easily ordered on your next primer order.