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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Real Time PCR
Posted by: Denae (IP Hidden, New member, 0)
Date: June 23, 2004 11:03AM
Hello All, Currently I am working on a real time pcr protocol that works in the UK (same primers and probes) but refuses to work in my lab (US). The issues are that amplification is proceeding poorly. The geometric phase is not linear due to non-optimal amplification, and when trying to multiplex my endogenous control with my target the target amplification is completely disrupted. I have had luck getting them to amplify more proficiently by reducing probe concentration, but cannot seem to get the multiplex functioning. I am running on the ABI 7900 using TAMRA probes (ABI) I am attempting to amplify a Multidrug Resistance Gene in Malaria parasite Plasmodium falciparum from whole genome samples(AT rich genome). I am following ABI's recommended "Universal PCR conditions" and using their "Taqman Universal PCR Master Mix". Conditions are 96/5min-denature then 40 cycles of 96/15s and 60/1 min. I am unsure of where to begin, so reaction mixture as follows: 2 micro L template (.2ng - Pf genome much smaller than human so need much less template) 5 microL mix (standard 1X conc. at 1/2 rxn volume) 300 nM primer 100 nM probe fill to 10 microL with dH20 Melting temps for the primers range from 52-58. Have tried varying temps, but found that anything below 58 looks even more pitiful than at 60. If anyone has had similar difficulties, or can think of a fundamental mistake I could be making please let me know.
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