Agarose gel electrophoresis/Common issues: Difference between revisions
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There are a number of issues which plague gels far a variety of reasons. | 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 | 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: | Possible unexpected bands: | ||
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#Primer-dimers (how long?) | #Primer-dimers (how long?) | ||
#Empty site in BB vector (~300bp band) | #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. | |||
[[User:ClarkeS|Sean]] 11:32, 3 Aug 2005 (EDT) | |||
[[Image:PCR_X_var_Tm_and_template.jpg]] | |||
Revision as of 08:32, 3 August 2005
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:
- Supercoiled
- Nicked
- Does anyone have good examples of these?
- Primer-dimers (how long?)
- 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)
