Agarose gel electrophoresis/Common issues

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Revision as of 13:12, 25 August 2005 by Barry Canton (talk | contribs)
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There are a number of issues which plague gels far a variety of reasons. This page will contain some of the more common problems and show examples of gel images that have those problems.

So if you get a gel with something bizarre, keep the image and post it here so that others can learn from your trouble.

Possible unexpected bands:

  1. Supercoiled
  2. Nicked
    • Does anyone have good examples of these?
  3. Primer-dimers (how long?)
  4. Empty site in BB vector (~300bp band)

Can anyone explain this smearing? Is it nonspecific primer binding? Nuclease activity? The gel is the product of PCR to amplify clpX from a Sauer lab plasmid. The lanes are from left to right: 2 log ladder, 0.5 ng template, 5 ng, and 13 ng. The 13 ng seem to give the desired product at around 1275 bp. Should I go even higher in template mass? I'd appreciate any suggestions on optimizing primer-template ratio.

Sean 11:32, 3 Aug 2005 (EDT)

PCR X var Tm and template.jpg