RNase treatment - genomic DNA sample lost during purification - RNase treatment troubleshooting (Mar/14/2007 )
Here is the protocol I am using - but doesn't ammonium acetate precipitate DNA? so am I precipitating the DNA and RNase from the supernatent at step 4? Please any suggestions...!
1. Resuspend the ppted and dried gDNA in 300 microliters of 1X TE buffer.
2. Add 15 microliters of 1 microgram/microliter RNAse A. Incubate in a 65° C water bath for 10 min.
3. Add 0.4 vol (126 microliters) of 7.5 M ammonium acetate to precipitate the RNAse A, flick the tube several times to mix.
4. Let stand for 30 min. at 4° C, spin at 13,000 rpm for 30 min. at 4° C, and carefully remove supernatent to new tube. Save the supernatent. Be careful and use a pipette rather than pouring, as the pellet is not visible.
5. Precipitate DNA from supernatent with 1 ml of cold 100% ethanol, mix gently for 1 min. and let stand at -20° C for 30 min.
6. Spin at 13,000 rpm for 20 min. at 4° C, decant and discard ethanol.
7. Wash pellet with 100 microliters wash buffer for 5 min., spin for 5 min. at 13,000 rpm, carefully remove and discard wash buffer using pipette.
Wash pellet with 100 microliters cold 70% ethanol, spin for 5 min. at 13,000 rpm, carefully remove ethanol and allow pellet to air dry using the heat lamp (about 15 minutes or until the pellet appears dry. Usually it "disappears" when dry).
Resuspend the pellet in 20 to 30 microliters of 1X TE buffer.
no. the dna won't precipitate until you add alcohol.