Not getting your expected #PCR bands even with established primers or even for #housekeepinggenes? The evil might be lying just in your template DNA!
What went wrong?
a. Using a generalized DNA/ RNA extraction kit
b. No use of template storage buffer (Eg. Tris-EDTA)
Solutions?
1. Primarily,you can dilute your template DNA serially like 1:10 and 1:50 and 1:100, setup PCR, analyse the data and prove the presence of inhibitors if you get PCR bands at higher dilutions. In case of Real Time PCR, Quantifiler™ Human DNA Quantification Kit can be used to assess the presence of inhibitors as it is one of the most sensitive kits available currently for Real Time PCR.
2. The next step would be to identify what kind of PCR inhibitors might be present there, in case of Stool DNA, there is high chance of #HumicAcid being present. This will allow you to choose the right kind of DNA extraction kit specific for the kind of sample.
TABLE: Different kinds of #PCRinhibitors
4. Use decontaminating solutions wherever possible, like NALC-NaOH (N-acetyl-L-cysteine-sodium hydroxide)buffer.
5. Use Taq polymerases that are more tolerate to inhibitors like AmpliTaq Gold®DNA polymerase.
6. lastly you can use certain additives like #BSA which provides protection to your reaction from Heme etc.
Hope it helps!
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