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If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...

If I am doing PCR and gel electrophoresis of the amplified DNA, what likely... - PCR - Polymerase Chain Reaction Forum

If I am doing PCR and gel electrophoresis of the amplified DNA, what likely... - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 11-22-2011, 07:16 PM
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Default If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...



...went wrong with these results? 1) Every lane has a fuzzy band about 50 bp long

2) All the other lanes are perfect except 1 lane has only a fuzzy band about 50 bp long
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Old 11-23-2011, 06:58 PM
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Default Re: If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...

Could be a primer dimer
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Old 11-29-2011, 01:20 PM
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Default Re: If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...

@mmorgan how did you guess that? I'm a newbie and still use troubleshooting software tools. For problems like this they are not much of help.
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Old 12-03-2011, 02:33 PM
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Default Re: If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...

Comes with experience.

if it's a low band, below the 100 bp line and looks fuzzy - it's likely primer dimers. If you can upload a gel picture, perhaps we can offer more help.
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Old 12-03-2011, 03:22 PM
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Default Re: If I am doing PCR and gel electrophoresis of the amplified DNA, what likely...

Quote:
Originally Posted by Albet View Post
@mmorgan how did you guess that? I'm a newbie and still use troubleshooting software tools. For problems like this they are not much of help.
If the product is approximately 50 bp long it's likely that the 3' ends of the primers (which are usually 15-30 nt long) annealed together. When extension occurs, it generates a product of about 50 bp.

Most primer design programs will look for 3' complementarity as well as primer hairpin potential. I have used MacVector for primer design and it seems to be quite good. Many people swear by Primer3 which is available for free as a web interface.

@Sugar Magnolia:

Could you post the sequence of the primers you used??
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amplified , dna , electrophoresis , gel , pcr


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