Removal of RNA from DNA sample - (Nov/20/2009 )

ya you can very well use the isolated DNA to prepare the standard curve. that will lessen any errors due to the standard curve of a DNA without contamination if the contamination is your problem.
It is some times possible that at higher concentrations of template, it masks the background but at lesser conc it cannot due to low amount of template!!

Pradeep Iyer on Nov 23 2009, 12:28 PM said:

Thank you Pradeep for your replies.

I'll give some try to look at what really is wrong and probably will come to forum later.

Ya sure do that
BEst luck!!!

This may be rather naive but: what stops you from adding some more RNAse A to your sample?
I don't think that should interfere much with your PCR reaction..

warsel on Nov 24 2009, 06:00 PM said:

Hi Warsel

I tried with higher volume of RNA as well though I've understood the small quantity of RNase is also sufficient for the degradation of RNA. But the problem remains the same. Now I think the RNA-nucleotides are also precipitated during centrifugation and that gives smear on my gel. Can that be the problem? Any suggestions are appreciated.

Thanks

Hmm.. are you sure what you are looking at in that gel is RNA?
RNAse A is an incredibly stable enzyme - if incubating your purified sample with RNAse A again does not remove the smears I'd try to find out if it's sheared DNA..