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?
- 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
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.
Used Qiagen DNeasy Blood & Tissue Kits and purified the ear lysate sample that was prepared by TaqMan™ Sample-to-SNP™ Kit. The yield was low.
Also, tried different polymerase to PCR amplify from the cured ear lysate (not purified) only Terra PCR Direct Polymerase Mix worked well.
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