DNase treatment in RNA isolation - protocol for DNase treatment (Sep/08/2006 )
I have isolated total RNA through commercially available kit, but i found DNA contamination in my sample so now i want to give DNase treatment, i have RNase free DNase from genei (1u/ul), i need protocol of DNase treatment so friends plz help me.
Have you check the Net for a protocol? Although RNA is not my field, a DNAse digest sounds simple enough. Even the place where you purchased the DNAse should provide a basic protocol.
Nonetheless if you do want help, I am in the opinion that you should make a second inquiry at the RT-PCR forum. I may be wrong but I have the feeling this forum is mainly looked at by people working on DNA rather then RNA. (i am one of them I have 4 protocols to kill RNA with me. And everything to preserve DNA.)
Best of luck, hope you find your protocol.
In our lab, when we isolate total bacterial RNA for microarray, we usually do both on-column DNase treatment and off-column treatment. As for on-column treatment, we use Qiagen on-column DNase (you can check its protocol and catalogue on qiagen website). As for off-column treatment, we digest 50ug of RNA with RQ Dnase (Promega). Use 0.1unite per 1ug RNA. Incubate at 37C (on the dry-hot block, not waterbath), for 1hour. Then we move the enzyme and its buffer according to the RNA clean-up protocol in the RNeasy Mini Handbook (qiagen). Then we determine the concentration by Nanodrop and check DNA comtanimation via normal PCR.
I tried this 3-4 times, and it seems to work well! No DNA contamination!
hope it helps!
I use DNase I from Ambion. it is straightforward and easy to use. it works well with us.
I use qiagen kit (RNeasy mini kit) )for RNA isolation, it is provided with a simple protocol for on-column DNase digestion with the RNase-free DNase set.
by the way I remember some one in this furom recommand Dnase prepared from shrimp which can be totally inactive by heat treatment and donot affect follow RNA manuplation, only caution is single DNA might not digest..