Agarose gel electrophoresis/Common issues

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Current revision (21:19, 3 January 2006) (view source)
m (Common Agarose Gel Issues moved to Agarose gel electrophoresis/Common issues)
 
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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.
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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.
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So if you get a gel with something bizarre, keep the image and post it here so that others can learn from your mistake.
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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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#Supercoiled
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==Supercoiled==
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#Nicked
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==Nicked==
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#*Does anyone have good examples of these?
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#Primer-dimers (how long?)
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==Primer-dimers==
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#Empty site in BB vector (~300bp band)
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[[Image:P17101&I7102|200px]]
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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.
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==Empty site in BB vector (~300bp band)==
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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.
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[[User:ClarkeS|Sean]] 11:32, 3 Aug 2005 (EDT)
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[[Image:PCR_X_var_Tm_and_template.jpg|300px]]

Current revision

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:

Contents

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)

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