I used TaqMan DNA extraction reagent (Applied Biosystems™ TaqMan™ Sample-to-SNP™ Kit) to prepare the mouse ear clip to screen for targeted gene by TaqMan qPCR and found some positive clones. But I am unable to PCR amplify the DNA from the sample by normal PCR using polymerase like goTaq or platinum Taq. Even the control genes does not get amplified. I feel there might be something in the extraction reagent that is preventing the normal PCR. Is there a way to purify the sample to use it for normal PCR amplification?
Laurence Stuart Dawkins-Hall
Try taking tour genomic DNA sample and precipitating the DNA using 1/20 3M sodium acetate pH 4-5 and 3 volumes of ice cold ethanol @ -20C for 1 hour and then wash the pellet with room temp 70% ethanol before drying resupending and then repeating the PCR I would also try repeating your PCR with other primers that have been used successfully with other DNA samples and other DNA samples, successful in the past with your existing primers (that hitherto have failed to work) Finally when you perform genomic DNA amplification make sure you use 20-50nG of DNA
- Taq Man is generally > 10 x morer sensitive than Gel based PCR so occasionally you will detect bands by qPCR and not agarose gels
- Specifically, you can visualise amplicons on gels down to about 10ng but not below that whereas products with yields of 1ng are detectable reliably by Taq Man
- That said, it is however suprising that you cannot detect your control genes
- Assuming your reagents for PCR are not denatured - have you tried fresh aliquots of PCR reagents including dNTPs which do degrade with freeze thawing - the best advice I can give you is to
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Marcin Sikora
You can try Ampure or sellf made version of it to purify DNA. There are some PCR additives like BSA that bind inhibitors in the PCR reaction. Also in my case use of Phusion polymerase helped in case of some fly DNA samples.
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