I am trying to do ligation-mediated PCR, and I am a newby. I'm using a published protocol, and based on the author's materials and methods, I understand that my longer adaptor (25bp) binds the 4bp 3' overhang left by my restriction enzyme, then my shorter adaptor (21bp) binds to the longer adaptor (without the 3' overhang region). Then, this is subjected to PCR with primers in 1) a 5' known region and 2) a primer binding exactly (21bp) to the shorter adaptor. I guess my question is, why do I need two adaptors? Why couldn't the second adaptor be eliminated?
Thanks in advance!
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