RT-PCR gone bad - (Feb/02/2005 )
Well, this seems to be the place for PCR advice.
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?
change the water. cheapest thing to do. Do you run a negative control? are there bands in there too?
or try fiddling with the amount of template.
find the source of contamination.
does cDNA go off?
I second vetticus3.
Change the water (!) and get it straight from the source.
Ya might want to get rid of your pipet tipes (use barrier tips) and use a fresh batch of PCR tubes. If that doesn't work...you should change the location in which you set you samples up. Perhaps a nice clean hood in another room...you may have DNA contamination in you PCR prep area.
Think happy thoughts
Thanks guys. It turned out that the water had gone "off". I alos autoclaved new tubes.
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.