Getting rid of DNA template from in vitro transcription rxn - (Jan/16/2006 )
HI,
I need help on how to get rid of DNA template from RNA transcript (after RNA purifying) even after DNase treatment of 30-45 minutes.
Should I leave the DNase in for longer?
Thanks,
Phage
-Phage-
Hi,
How long are you running your DNase reactions? Also, what methods have you used for RNA purficiation?
Thanks,
Hank
-haringsh-
QUOTE (haringsh @ Jan 16 2006, 04:28 PM)
Hi,
How long are you running your DNase reactions? Also, what methods have you used for RNA purficiation?
Thanks,
Hank
How long are you running your DNase reactions? Also, what methods have you used for RNA purficiation?
Thanks,
Hank
RNA purification is via a purification kit (stratagene) and DNase treatment is for about 45 minutes or so.
Thanks,
Phage
-Phage-
Me again on this topic ...
Did you try several DNA'ses and made sure it works on a template to be sure DNA? Did you add an RNAse A for an aliquot just to make sure it really is DNA?
-wincel-
QUOTE (wincel @ Jan 17 2006, 04:49 AM)
Me again on this topic ...
Did you try several DNA'ses and made sure it works on a template to be sure DNA? Did you add an RNAse A for an aliquot just to make sure it really is DNA?
Did you try several DNA'ses and made sure it works on a template to be sure DNA? Did you add an RNAse A for an aliquot just to make sure it really is DNA?
I've not tried several DNases.. just one and that may be from a pretty old batch too. So i'm going to repeat it with a fresher one.
-Phage-
QUOTE (Phage @ Jan 18 2006, 05:00 PM)
QUOTE (wincel @ Jan 17 2006, 04:49 AM)
Me again on this topic ...
Did you try several DNA'ses and made sure it works on a template to be sure DNA? Did you add an RNAse A for an aliquot just to make sure it really is DNA?
I've not tried several DNases.. just one and that may be from a pretty old batch too. So i'm going to repeat it with a fresher one.
On further note: there is a paper dealing with DNA-contaminations from Mycobacteria in biomed central. They found that two times 20 seconds sonification 0.9seconds intervall with 20% of power before DNAse digestion gives much better DNA-digestion probably by dissolving secondary structures of the DNA. You could try this too maybe. They had performed those experiments to get rid of RT-PCR false negative bands from still existing DNA contamination.
-wincel-
QUOTE (Phage @ Jan 16 2006, 01:51 PM)
HI,
I need help on how to get rid of DNA template from RNA transcript (after RNA purifying) even after DNase treatment of 30-45 minutes.
Should I leave the DNase in for longer?
Thanks,
Phage
I need help on how to get rid of DNA template from RNA transcript (after RNA purifying) even after DNase treatment of 30-45 minutes.
Should I leave the DNase in for longer?
Thanks,
Phage
I add my DNase in for 20 minutes and that is enough.... you must have something in your solution that is inhibiting your DNAse... try either an quick initial purification (pcr prep qiagen or other) and then add the Dnase... 37C for 30 minutes several Units of DNase should eat up all the DNA even several ugs worth in no time
cheers
-aristotle-