gene was amplified when -RT were used as template? - (Dec/06/2007 )
hi, all
as you know, -RT was used to detect the genomic contamination. today, when I used -RT template to amplify GAPDH as a negative control, a band which had same length with RT template was detected, although it was much slighter. I'm sure the primers I used are intron-spanning. If there were genomic contamination, a bigger band would be detected, but I didn't find a such one.
anyone kindly tell me why?
Could a gene be amplified when using RNA as template?
Thanks!
Hi,
The most probable explanation is that you got contamination in your -RT, either from cDNA or from PCR product. How does your non template control (water) look like?
as you know, -RT was used to detect the genomic contamination. today, when I used -RT template to amplify GAPDH as a negative control, a band which had same length with RT template was detected, although it was much slighter. I'm sure the primers I used are intron-spanning. If there were genomic contamination, a bigger band would be detected, but I didn't find a such one.
anyone kindly tell me why?
Could a gene be amplified when using RNA as template?
Thanks!
I'll say check your primers to confirm the span an intron, you can try a PCR with gDNA if you have any available. If you are getting PCR product from -RT samples, your most than surely have DNA contamination, sorry.
you've got plain old contamination.
some of your cDNA was transfered over. either in primers, water buffer, etc etc.
V
my primers or water may be contaminated. I did a negative control with water, and a band same with -RT appeared.
there are two bands with genomic DNA, one is same with RT, and the other is amplified from hela genomic DNA.
but the former is much brighter than the latter, why?