RNA isolation - DNA contamination troubles (Jul/14/2005 )
What is the best way to remove DNA contamination after RNA isolation from cells after culture? Does anybody have protocol how to use DNase in this case? Follow the RNA isolation or during isolation?
there are two way to do a DNase teartment. some companies offer DNase together with their RNA cleanup kits, and you can follow the manufacturers protocol for a on column digestion, meaning you are DNaseing the RNA in the purification process.
the other way is to treat the completely purified RNA later with DNase, which can be from any brand. usually supplied with it's own buffer, this is what I do and what worked best for me... I got reduction of residual DNA of about 99,95% as determined by qPCR. you should mind that no enzymatic reaction is ever 100% efficient, so some DNA molecules always "survive" .....
I have similar problems with DNA contamination and I am considering the use of DNase. I am afraid that inactivation of DNase wouldnt be complete, so it could degrade cDNA. Besides I wonder if DNase I buffer is compatible with AMV reverse transcriptase.
I have done RNA preparation from mouse tissue, then make RT and then make PCR.
94 oC 2 min
94 oC 15 sec
61 oC 30 sec
68 oC 2 min 20 sec
I've used Pfx polymerase from Invitrogene... But I don't have band of DNA of interest, but I have very very big band of DNA of a very big size... It doesn't really go through 1% Agarose gel... So it's not far away from pockets... 30-40 kB??? But the band is pretty nice, I mean if the size was correct (2,3 kB) I was really happy about so nice band...
What do you think could it be? Also DNA contamination of my sample of RNA, or there are some other theories about it?
Methylnick suggested in this topic that Turbo DNase from Ambion is good. Surfing the forum, i found other user that did use it (here).
And for end, there was a little discussion here regarding DNAse treatment on RNA smaples.
I hope that gonna help.
Nothing other than DNAse treatment will remove all DNA from an RNA prep.
One useful tip is to use Mn2+ instead of Mg2+ in the DNase I digestion buffer. Mn causes DNAse I to make double stranded cuts rather than DNA nicks (DNA with nicks on one strand only can still be PCR amplified).
Also, you can do a acid phenol:chloroform:isoamyl extraction (pH ~4.7). DNA will go into the organic phase and RNA into the aqueous. Using this in combination with the previously suggest DNase treatments should work out for you.