Sv: Problem: removal of DNA from RT-PCR using DNase
ali at image.dk
Tue Sep 12 13:12:30 EST 2000
Try tjecking your cDNA for DNA contamination with an intronspanning
primerset (should be negative!). I have experienced that my no-RT control
had a very faint band no matter how long time i DNAse treated my sample. I
found that my PCR polymerase had RT-activity.
Also remember to run a watercontrol as your reagents might be contaminated
<teresa_mogul at my-deja.com> skrev i en
nyhedsmeddelelse:8pk1bq$u57$1 at nnrp1.deja.com...
> I have just begun RT-PCR experiments. They are going very well so far
> with beautiful amplification as show on agarose gel. However, when I run
> the no-RT control I have a very faint band indicating that I have not
> fully removed the DNA from my RNA. I measured the amount of RNA/DNA in
> my sample and according to the manifacturers notes 30 minutes at 37°C
> with 1U DNase should be ample to remove my DNA. Increasing the time does
> not seem to help. Has anyone got any suggestions? I am using glass
> microfibre plates and guanidinium thiosyanate for extraction could there
> be a DNase inhibitor in my DNA/RNA extract?
> Thanks in advance for any help
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