How to know which primer/s work?

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magicsiew
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How to know which primer/s work?

Post by magicsiew » Fri Aug 27, 2010 1:45 pm

Hi, I have designed 4 pairs of degenerate primers to amplify a gene for cloning purpose. The primers are just trial and error as they are designed based on related organism. I have ordered the primers but not yet gotten and testing it. What I want to to ask is, how will the gel picture looks like if only forward primer get to target and start to amplify the gene, while reverse primer fail to do so, and vice versa? Let say, if my set 1 forward primer works, and set 3 reverse primer works, other all doesnt work, how is the gel pic will look like?

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canalon
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Post by canalon » Fri Aug 27, 2010 4:23 pm

If only one primer works, the amplification will not be exponential and the total amount of DNA synthesized will be too small to be detected. So in short, your gel will look empty. Only solution in this case is to try PCRs with all the possible combination of primer pairs that might yield a result.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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JackBean
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Post by JackBean » Fri Aug 27, 2010 4:37 pm

well, it won't be really empty, but there will be only smear, because the amplified pieces of DNA
1) will be in low amount
2) will be of different length
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

magicsiew
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Post by magicsiew » Fri Aug 27, 2010 6:00 pm

Thanks you guys for the information.

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