contamination in real-time pcr negative control - (Mar/11/2005 )
I had this trouble too, and sometimes still happens but after 35-40 cycles. If You get a signal in negative control under the 35-30 cycles You're having troubles. If You run triplicates and do not get results in all the three tubes I would exclude the primer working-solution contamination. More plausible is the possible carryover from the pipet. As someone already said: try to perform the reaction in a clean zone and, if possible, separete the negative control tubes from the rest [ie use a strip specifically for the blank and keep it far from the others].
I hope this could help.
I hope this could help.
After reading much on this topic, I tried using more controls to test the source...
1- no RNA
2- no RT
3- no cDNA
4- no primers, just amplicon used in curve
5- just water
the no primers and just water were totally negative, which elimnated carryover and Sybr green as sources. The others showed up late in the cycles, and not all of them amplified in all of the triplicate wells for all of the genes...some genes were clean...which leads me to think primer dimers?
Any thoughts?
Run NT with " carrier " nucleic acid
tRNA can help
"empty" NTs only help Taq to find "something" to amplify 
))
although Taq can perform so called "primer-template independent" sythesis )
Take dNTP + Taq and put it for 2-3 hours at 70 C
You will get tons of DNA on gel
And some Taqs from some companies are extremelly " effective " in making
"de novo " DNA without primers and templates