RT-PCR gone bad
Posted 02 February 2005 - 04:39 PM
I am amplifying cDNA. I previously amplified my samples and got one
discrete band as expected. This method worked 3 times. However, when I do the PCR now I get ~3-7 bands. I have changed, dntps, and my primers. The bands also appear with my samples that previously gave me a clear result. Any suggestions?
Posted 02 February 2005 - 04:59 PM
or try fiddling with the amount of template.
find the source of contamination.
does cDNA go off?
Edited by vetticus3, 02 February 2005 - 05:00 PM.
Posted 03 February 2005 - 04:30 PM
Change the water (!) and get it straight from the source.
Posted 03 February 2005 - 06:17 PM
Think happy thoughts
Posted 04 February 2005 - 05:25 PM
I had done water controls which showed no bands. But when I did the PCR side by side with new water the one with new H2O had one band while the old one had multiple bands. Maybe the bottle in which the water was autoclaved was contaminated.
AND the best advice of all is the hardest to follow but the most important: Think happy thoughts.