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No bands for PCR product on agarose gel

No bands for PCR product on agarose gel - PCR - Polymerase Chain Reaction Forum

No bands for PCR product on agarose gel - 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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  #1  
Old 10-24-2011, 10:44 PM
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Default No bands for PCR product on agarose gel



Hi,

I've been trying to amplify a gene (IFT52) using PCR and then running the product on agarose gel but the last 3 times I've tried no bands show up.

The PCR mix I'm using is:
sterile water: 30ul
polymerase buffer: 10ul
dNTP: 5ul
forward primer: 2ul
reverse primer: 2ul
cDNA template: 0.5ul
Taq polymerase: 0.5ul

Once the PCR is performed the following different concentrations of the PCR products are loaded into the gel
PCR product 1ul + sterile water 7ul + 2ul loading dye
PCR product 5ul + sterile water 3ul + 2ul loading dye

I don't know why I don't seem to be getting any bands on the gel. I've changed the cDNA source and still nothing is coming up. Any help on what might be going wrong or is possibly going wrong I'd really appreciate the feedback.
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Old 11-02-2011, 02:15 PM
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Default Re: No bands for PCR product on agarose gel

are you running a positive control as well? If so, is it showing up on the gel?
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Old 11-03-2011, 12:06 PM
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Default Re: No bands for PCR product on agarose gel

Yes as Saxatilis mentioned you have to perform the pcr using appropriate Positive control.
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Old 12-10-2011, 05:41 AM
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Default Re: No bands for PCR product on agarose gel

the concentration of PCR product with loading dye is too litle
mahmoud manama
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Old 12-12-2011, 04:24 PM
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Default Re: No bands for PCR product on agarose gel

Also, make sure that the gel concentration (i.e. 1% agarose 1.5% agarose, etc) is appropriate for the size of your bands
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Old 12-14-2011, 03:40 PM
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Default Re: No bands for PCR product on agarose gel

cDNA template: 0.5ul
please, incras the volume for 1 ul
mahmoud manama
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