This protocol will get rid of DNA in an RNA sample, when you want to do RT-PCR on the RNA without having any DNA contamination
- Two different methods, depending upon whether you have normal or severe DNA contamination. (Severe can occur in certain tissues like spleen, kidney and thymus). The following protocol is for normal DNA contamination (up to 50 µg/ml RNA).
- Normally done in a 50 µl reaction (10 to 100 µl) with up to 10 µg of RNA
- Add 0.1 volume 10X turbo Dnase buffer and 1 µl Turbo Dnas to the RNA and mix gently. Incubate 37ºC for 20-30 min.
- Add 0.1 volume (or 2 µl, whichever is greater) resuspended (vortexed) Dnase Inactivation Reagent and mix well. Incubate 2-3 minutes room temp, mixing three times during incubation.
- Centrifuge 10,000 g for 1.5 min. transfer RNA to a clean tube.
- If you started with 10 µg, can expect to get ~7 µg at the end, 160 ng/µl in 45 µl.
DNase reaction