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Pls help: Troubleshoot Taqman Real-Time PCR

Pls help: Troubleshoot Taqman Real-Time PCR - Real-Time PCR and Quantitative PCR Forum

Pls help: Troubleshoot Taqman Real-Time PCR - Real-Time PCR and Quantitative PCR Forum


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Old 01-13-2011, 04:32 AM
Pipette Filler
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Default Pls help: Troubleshoot Taqman Real-Time PCR



I have a doubt regarding taqman real time PCR. Last friday I reconstituted new taqman probes (FAM BHQ 1 and Cy5 BHQ2) and primers into 100 uM of concentration. and i ran the taqman real time assay and got nice melting curves. Then on Monday i continued the assay with some more samples. But starting from Monday till now i don't seem to get any amplification/result. The curves look pretty messy. Basically no amplification observed. The same thing happened few months ago when I ordered another batch of probes an primers. I ordered from NHK bioscience. I suspect the probes are degrading fast once reconstituted. Is it possible to degrade within 3 days? I have tested with new aliquotes of probes, Primers and all reagents and also new aliquot of positive crtl.Even positive ctrl not showing up. Any idea?

Thanks for sharing.
Regards,
Jeyanthi
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Old 02-15-2011, 02:06 AM
Pipette Filler
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Default Re: Pls help: Troubleshoot Taqman Real-Time PCR

do you have a screen shot of what your results look like? What did you hydrate your probes with?
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