Agarose gel electrophoresis/Common issues: Difference between revisions
Barry Canton (talk | contribs) No edit summary |
Barry Canton (talk | contribs) No edit summary |
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Possible unexpected bands: | Possible unexpected bands: | ||
==Supercoiled== | |||
==Nicked== | |||
==Primer-dimers== | |||
[[Image:P17101&I7102|200px]] | |||
The bright fuzzy bands at the bottom of the gel are typical of those caused by primer dimers. Primer-dimers can be minimized by using hot start PCR or by using a higher annealing temperature. Preventative measures include [[Designing primers|designing primers]] that do not form homo- or hetero-dimers. | |||
==Empty site in BB vector (~300bp band)== | |||
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[[User:ClarkeS|Sean]] 11:32, 3 Aug 2005 (EDT) | [[User:ClarkeS|Sean]] 11:32, 3 Aug 2005 (EDT) | ||
[[Image:PCR_X_var_Tm_and_template.jpg| | [[Image:PCR_X_var_Tm_and_template.jpg|300px]] | ||
Revision as of 14:20, 25 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
Primer-dimers
The bright fuzzy bands at the bottom of the gel are typical of those caused by primer dimers. Primer-dimers can be minimized by using hot start PCR or by using a higher annealing temperature. Preventative measures include designing primers that do not form homo- or hetero-dimers.
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)
