Hi, does anyone can give a suggestion?
I have a RFLP result but seems faint. By following manufacturer's procedure, the 10ul amplicon diluted with 18ul NFW, 2ul 10x Tango buffer and 1ul enzyme. Means that the amplicon concentration is lowered into it's half. The bands are faint especially for low base pairs (about 60bp).
I am curious, is it possible to decrease or even replace all nuclease free water with PCR amplicon, but still consider the tango buffer concentration and enzyme? Thank you!
With many enzymes you can simply cut in pcr buffer. Check the New England Biolabs web site for a list of how well many enzymes cut in PCR buffer. If you are using a lot of pcr product then add the RE buffer you can have difficulties if the enzyme needs a low salt buffer but the mix of pcr buffer and RE buffer adds up to high salt. With some rare cutter enzymes you may need to use more enzyme than expected as the activity is measured in cuts which are far apart in genomic dna but every short amplimer from pcr may contain a cut site
10ul amplicon diluted with .......
>> This is not a logical way to talk about DNA. Volume of DNA/amplicon does not mean anything without its concentration.
If the DNA concentration was low, dilution reduced it even further causing the faint band, as you describe it.
Follow the protocol according to amount/concentration of DNA and not volume.
Thank you @Paul Rutland , I have read your suggestion in a previous question about effect of too much salt on restriction enzyme reaction. The consequences are low efficiency and the result of electrophoresis.
Thank you @Abhijeet Singh it was refer to manufacturer's protocol, they just mention the volume. Off course it caused by the low quantity of pcr products, that's why i looked for suggestion if NFW replaced with pcr product.
I am facing the same problem. can you tell me what to do? Should I reduce the volume of nuclease-free water?
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