primer/probe mix for standard generation? - (Jan/15/2008 )
do you think a primer/probe mix (with VIC dye label, minor groove binder (MGB) and non-fluorescent quencher (NFQ), Applied Biosystems) can be used for initial generation of PCR products and subsequent cloning to generate standards for quantitative PCR? I wonder if the probe or fluorescence of the primers may hinder this process? As I do not know the sequence of primers used in the mix supplied by Applied Biosystems it seems convenient to just use the mix.
you can amplify it with conventional PCR. your enzyme just needs the 5'-3' exonuclease activity.
but i would recommend to simply use the purified PCR products as (relative) standards. this is much simpler and faster than to clone them into a vector. I also have to admit that in my case cloning into a vector never worked with a PCR from an ABI primer/probe mix. only with conventional PCR products. my only explanation is, that there is some ingredient sticking to the DNA (i purified with silica gel columns, with gel, ...) which inhibits the ligase reaction. I tried blunt-end cloning into fermentas pJET vector (yes, i blunted the pcr-product). Supplied control PCR product worked, my own PCR products worked, just the products from the qPCR-mix didn't.
ABI is doing the same by their own, just read here:
Thanks a lot.
The vector method works with primer/probe mix from Applied Biosystems when using purified (GFX gel purification) or unpurified PCR product with TOPO-TA kit (where ligase is an inherent property of the pCR2.1-TOPO vector). I had done this and retrospectively wonder what may have happened to the probe during the process and if for any reason the primer/probe mix instead of a conventional primer pair may influence results. However as fluorescence is restricted to the probe, not primers, I guess the method should be valid.