How to know which primer/s work? - Biology Forum

last updated by

magicsiew 14 years, 2 months ago

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- August 27, 2010 at 1:45 pm #13701
  magicsiew
  Participant
  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?
- August 27, 2010 at 4:23 pm #101040
  canalon
  Participant
  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.
- August 27, 2010 at 4:37 pm #101041
  JackBean
  Participant
  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
- August 27, 2010 at 6:00 pm #101043
  magicsiew
  Participant
  Thanks you guys for the information.
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