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amplification in negative control in RT PCR - (Jun/08/2009 )

:D dear friends

I have facing a serious problem with the amplificaton in negative control (without C-DNA) in my real time experiment.
all time i got amplifcation after 35 cycles and some time it my be larger than my amples.
so not able to differentiate between my sample amplification and nonspecific one.
i have roche 2.0 light cycler capliiary based system.

-Gaurav-

You have contamination in your PCR system: water, primers, dNTP, polymerase, etc. You need to find out which components are contamination or discard everything.

-pcrman-

It's contamination...
Try to check if your primers are not contaminated.
One suggestion:
CHANGE YOUR BENCH AND repeat at a new place... Sometimes, there is RNASe, all sorts of pcr product contamination which cannot be seen....
this sounds funny..but it solved my problem once...then i cleaned my bench thoroughly with bleach and washing liquid...before coming back to my bench...


Gaurav on Jun 8 2009, 10:10 PM said:

;) dear friends

I have facing a serious problem with the amplificaton in negative control (without C-DNA) in my real time experiment.
all time i got amplifcation after 35 cycles and some time it my be larger than my amples.
so not able to differentiate between my sample amplification and nonspecific one.
i have roche 2.0 light cycler capliiary based system.

-kedar-