Strange results with DNase - any explanation? - (Jun/13/2008 )

Hello experts,

I am using DNase (RNase free) from NEB to remove all DNA template from invitro transcription reaction (done using SP6 polymerase).
Digestion done at 37C.
When the DNA intensity is followed in a time course on an agarose gel

a) for the first 20 minutes the DNA band intensity goes down.
however, the DNA band intensity again starts building up between 20minutes to 60minutes.

I am wondering what is going on.

Anyone has faced similar issues?

thanks.

Hello experts,

I am using DNase (RNase free) from NEB to remove all DNA template from invitro transcription reaction (done using SP6 polymerase).
Digestion done at 37C.
When the DNA intensity is followed in a time course on an agarose gel

a) for the first 20 minutes the DNA band intensity goes down.
however, the DNA band intensity again starts building up between 20minutes to 60minutes.

I am wondering what is going on.

Anyone has faced similar issues?

thanks.

Hi Patty

I have repeated the experiment twice. It is not an artifact.

This is because, I keep taking out samples from the same mix at different time intervals.

From 0min to 20min there is a decrease in the band intensity of the DNA band. However, after that the band intensity again starts increasing.

thanks.

Hmmm. THat's weird.

I wonder if it's due to something unusual. For example, maybe the DNAse isn't active, but is still able to bind to the DNA. Maybe the DNAse is binding to the DNA, which is interfering with gel migration and giving the appearance of reduced band intensity. Maybe after a while the DNAse can't even bind, and now the DNA can run through the gel properly again. can you post an image?

I have a hard time thinking that the DNA is actually being degraded and then re-polymerized. Not only because you aren't adding DNA polymerase, but also because you aren't adding dNTPs or primers.

I'd order fresh DNAse from a different company and try it. I'd also quantitate the DNA at different time points in a different way - say through qPCR - Being sure to inactivate the DNAse first.