Primer-dimer, non-specific binding, and other pcr issues

Today, we ran the gel for some of the seastar and Laby PCRs.  In spite of having rerun the PCRs, we got the same results.  The seastar primers seem to bind to non-specific regions and form primer-dimers.

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The primer-dimer is the lowest band on this gel.  Primer-dimers are formed by the two primers annealing to each other.  Unfortunately, it was difficult to distinguish from the intended product as we were trying to amplify a 100bp segment, which would be slightly above where the primer-dimer is.  We can also see some non-specific binding in the larger bands.  This can sometimes be fixed by raising the annealing temperature, but in our instance where we weren’t amplifying the intended product, seems to be a sign that we need to reexamine our protocol.  Hopefully we’ll have better luck next time!!

If you’re interested in reading more about troubleshooting PCRs, this guide is very helpful:

http://palumbi.stanford.edu/SimpleFoolsMaster.pdf

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