1 reply to this topic

[Shahin]

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?
Thanks
Shahin

[Trof]

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)