Hi!
I've been trying to decipher what happened to some of our runs with early amplification in the NTC and NEC. This has happened to us twice now. For background, we are using a mastermix of PCR components and probes which has been optimized before.
Any insights?
I have attached the graph and the one encircled are all from the NTC and NEC
Try using a different master mix, with fresh working stocks of Primers and Probes and see if the problem still persists. Also change your Nuclease Free Water, as it may also be a source of carry over contamination. For future qPCR work try using master mixes with UNG to stop carry over contamination. Also you can check by agarose gel electrophoresis of the NTC and NEC wells for PCR bands.
Hello
In those 3 samples you have encircled, there is amplification only until cycle ~8! Seems that absent of template, could result in formation of dimer/cross dimmer. To see what happened, you need to check the melt curve and agarose electrophoresis of some samples.
Try using a different master mix, with fresh working stocks of Primers and Probes and see if the problem still persists. Also change your Nuclease Free Water, as it may also be a source of carry over contamination. For future qPCR work try using master mixes with UNG to stop carry over contamination. Also you can check by agarose gel electrophoresis of the NTC and NEC wells for PCR bands.
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