I've been given a protocol for gDNA extraction to try out:
1) Add 500ul of CTAB buffer to 100mg of frozen ground mycelium & vortex to get rid of lumps, if there are still lumps add 10-15 1mm glass beads and vortex for 45 seconds at max speed.
2) Incubate at 65 degrees in a water bath for 40 minutes.
3) Add 500ul of phenol:chloroform:isoamyl alcohol (25:24:1) and mix until homogenous.
4) Centrifuge at 4000 rpm for 10 minutes.
5) Remove top layer to new tube & add 700ul of cold-isopropanol, invert once to mix.
6) Put tubes at -20 degrees for 2 hours.
7) Centrifuge tubes at 13,000rpm for 20-30 minutes.
8) Tip the supernatant off and ass 100ul of cool 70% ethanol to wash pellet. Centrifuge briefly to pool contents & tip off ethanol.
9) Repeat number 8 and then centrifuge again, then use a gilson to remove as much ethanol as possible - avoiding the pellet.
10) Air dry, then suspend pellet in 100ul of TE buffer. Store at -20 degrees.
My problem with this is it needs an RNase Step since gel electrophoresis revealed RNA contamination. However I need to alter the steps as little as possible. I've seen some methods use a a phenol:chloroform:isoamyl alcholol step, followed by isopropanol precipitation and then suspension in TE buffer and RNaseA, incubated at 37 degrees. Then another phenol:chloroform:isoamyl alcholol step is required and another precipitation.
I've seen some methods just add RNaseA at the end when the pellet is suspended in TE buffer, incubated at 37 degrees and then stored. But I'd rather not have RNase A in my final stage as I've heard some people blame it for poor PCR results, although some have said it's not the RNase that's to blame.
Is it possible to add it to the CTAB buffer stage i.e. after incubating the sample in CTAB at 65 degrees, could I add RNase A and then incubate at 37 degrees for 30 minutes?
The downstream application is amplification of microsatellites using primers with some redundant sites. I'm not 100% sure RNA should even affect PCR, but again for one page which says it's not a problem (A QIAGEN info sheet on gDNA) I see someone else claiming it is.
Edited by Axolotl9250, 15 August 2012 - 08:19 AM.