PCR contamination in NoRT Control - I need advice (Feb/26/2007 )
I am trying to do a RT-PCR for a serie of sequences of interest. Some of them didn't show a band with 30cycles but they did with 40cycles, a faint band.
The problem is that when I run my No-RT PCR sample I got a faint band in some cases but not others. Primers are intron-spanning but the band is still there and the same size that the expected one. In addition, for beta-actin, the band appears also in water control with 40cycles (not with 30cycles).. I believe that all this is because of aerosols and the dirt in the surronding areas and clothes. Ive been reading and following all pieces of advice about using different areas, changing lab coats. Ive already tried different primers for beta-actin, new purchased reagents, etc. My immortal betaActin band is there.
I must say that post and pre PCR areas are only 1.5m away.
Any suggestions? No clue about what else I can do to not get contamination with beta actin.
you should check for DNA contamination, even though you have used intron spanning primers, there are pseudogenes in the genome for many genes (esp. housekeeping genes such as beta actin) these pseudogenes do not have introns as they are integrated into DNA from the spliced mRNA version of the gene...