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| PCR - Polymerase Chain Reaction Forum 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
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| 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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#2
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| are you running a positive control as well? If so, is it showing up on the gel? |
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#3
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| Yes as Saxatilis mentioned you have to perform the pcr using appropriate Positive control. |
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#4
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| the concentration of PCR product with loading dye is too litle mahmoud manama |
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#5
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| 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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#6
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| cDNA template: 0.5ul please, incras the volume for 1 ul mahmoud manama |
| Tags |
| agarose , bands , gel , no bands , pcr , product |
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