real-time PCR with with DNA

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nin1318
nin1318's picture
real-time PCR with with DNA

so i am stuck...i would like to do quantitative PCR rather than qualitative, and have been unable to get the quant. to work.

in theory, there should be no difference, except for the addition of the fluorescent dye to the mix for teh real-time.

for the regular PCR i use amplitaq gold. for real-time, i use AB's 2X sybr master mix. the regular PCR works fine, it's never a problem. but i tried moving on to the real-time and i get crazy results, with lines all over the place when i try to look at the amplification curves (it's an abi7300 machine). i tried using new samples in case that was the problem, i tried using just total genomic DNA, same craziness. i ran the samples out on a gel, and not suprisingly, no band.

any suggestions?????

COHscientist
COHscientist's picture
did you try different melting

did you try different melting temperature?
I am not an expert, but I heard the Tm is kindof critical in Real time PCR.

nin1318
nin1318's picture
well i have the pcr optimized

well i have the pcr optimized already. it should work exactly the same, since it's a thermal cycler. the only difference should be that the machine records the fluorescence.

i ran the products on a gel and there are (not suprisingly) no bands.

nin1318
nin1318's picture
i'm still troubleshooting

i'm still troubleshooting with no luck so far...i've tried different cycling conditions, and a wide range of template concentration(1ng-100ng). it may be the actual master mix is bad-the applied biosystems tech support thinks this might be the problem. hopefully with a new sample of there new super sybr (or whatever it's called) i will get some results...