Genomic DNA Contamination of Real Time PCR - (Jan/04/2006 )

Hi all --
I'm amplifying 10ug to 1fg of bacterial RNA (DNase I on-column digested) followed by RT and Real Time PCR. I see genomic DNA (signal) in my No Amplifcation Control (NAC) samples. Has anyone else seen this phenomemon or been able to get rid of the gDNA? Thanks!

RBart

Hi,
I see your question was from a couple of weeks ago.
In case you are still interested in an answer, I may have a similar problem of gDNA contamination and would also like to know the best way to get rid of it! If anyone has any insights, please let us know.

Are you doing a dilution series?

cheers

quote name='RBart' date='Jan 5 2006, 03:26 PM' post='36079']
Hi all --
I'm amplifying 10ug to 1fg of bacterial RNA (DNase I on-column digested) followed by RT and Real Time PCR. I see genomic DNA (signal) in my No Amplifcation Control (NAC) samples. Has anyone else seen this phenomemon or been able to get rid of the gDNA? Thanks!

RBart
[/quote]

I am not sure which kit you are using, but I like Ambion DNAfree -- slurry allows you to remove the DNAse and nucleotides after digestion... With this kit, I would DNAse, use the slurry to clean up then repeat DNAse step... Could do similar with phenol chloroform extraction and EtOH ppt between, don't know if your columns would do something similar...

It just sounds to me like there is more DNA than the DNAse step can handle and repeating should be efficient enough to remove the detectable contamination... Other options are to reduce the amount you are DNAsing or increase the enzyme concentration, but I don't think you want to put too much enzyme in one reaction, (would be hard to get rid of) which is why I suggest doing two sequential DNAses...

HTH...

I had the same problem with the on-column digestion with Qiagen. I tried Amersham as well; I had the same issue.

I then switched to Ambion for Dnase (DNA free, I think is what they call it).

I no longer have a problem with it.

Best of luck,

Matt

[quote name='smurray' date='Jan 23 2006, 05:55 PM' post='38246']
Hi,
I see your question was from a couple of weeks ago.
In case you are still interested in an answer, I may have a similar problem of gDNA contamination and would also like to know the best way to get rid of it! If anyone has any insights, please let us know.

Are you doing a dilution series?

cheers

quote name='RBart' date='Jan 5 2006, 03:26 PM' post='36079']
Hi all --
I'm amplifying 10ug to 1fg of bacterial RNA (DNase I on-column digested) followed by RT and Real Time PCR. I see genomic DNA (signal) in my No Amplifcation Control (NAC) samples. Has anyone else seen this phenomemon or been able to get rid of the gDNA? Thanks!

RBart
[/quote]
[/quote]
The best way to get rid of the DNA is to use a heat labile double stranded specific DNAse, like Shrimp nuclease. It's heat inactivated, and works in most common beffers.
Then you do not need to worry about how to get rid of the DNase after use.
Very convenient, and in case you worry about if it's RNAse free; I do not think it's marketed as such, - but in our lab the RNase free DNaseI we have tested, shows more RNA degradation than Shrimp nuclease.