false positive/cross reaction with TaqMan - (Sep/27/2011 )
Hi every one
I have been trying to set up few TaqMan qPCR/Tests on RotorGeneQ and experienced following problems. please comment, thanks
1) for two of the tests, first time I set up the test, they were fine. but now about few months later. when I prepared new qPCR mixes, they show false positivity with water or Human genomic DNA. I use Fermentas probe master mix. I have resuspended probes and primers in ddwater and store at -20C.
2) some mixes work fine on RotorGene Q/6000 but the same mix shows false positivity on Step-One cycler. Do you have any idea what should be different in the same mix for RotorGene and ABI or plate format machines?
1) can be contaminated mastermix, try new batch of water, primers, probes and mix
2) Step One uses tubes, do you use separate tubes or plates? You could cross-contaminated the negative control if don't take care about proper handling the plate (avoid moving samples around other wells, little drops and purges of the pipett over the plate)