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PCR with one lone primer and another short

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PCR with one lone primer and another short

Postby rama12chandra » Mon Jul 18, 2011 3:09 pm

Hiii all ,
I want to do PCR with one long 130mer Reverse primer and another short 30mer forward primer.
I need to fuse His and V5 Tag to my protein, so i have designed those tagged sequences in my reverse primer to amplify the ORF with tagged seqence and later clone it into a vector.
Can anyone please suggest how to proceed with such primer or whether my approach is wrong...

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Postby JackBean » Mon Jul 18, 2011 4:32 pm

30mer is short? :lol:

Anyway, so that 130-nt primer contains the tag? It does not align whole, right? Cannot you amplify it from somewhere else?

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Postby canalon » Mon Jul 18, 2011 4:37 pm

Ignore the addition and design you program as if you only had the overlapping sequences and run a normal PCR. A 30 mer overlap is quite long, but why not, too long is not a problem, at worst a waste of (a little bit of) money.

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