I have optimized the annealing temperature from 50-47 degress. I have also optimized the volume of DNA added from 1-5 ul but the PCR products are still stuck in the well.
Rakesh Ganji
need more details, like, amount and source of template, expected size of your pcr product, percent of gel you have used.
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Balaraman Senthil Kumar
As rakesh says we need more info, as u say your product is stuck to the well reduce your gel percentage and run............as you cannot say whether the well has your PCR product or its dimer formation based on your observation.
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Lochlan Fennell
Could be due to a number of reasons:
- High % gel provides a greater resistance for the PCR product and hence it moves more slowly, if at all.
- Little run time to allow the product to move through the gel
- Low voltage - DNA is attracted to the positive end of the gel and hence a low voltage will yield a low attractive force and may trap the product.
Without knowing your exact situation I would suggest decreasing the gel concentration and increasing the voltage.
Do you see the (with the naked eye) the dye move through the gel at all?
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