Fungal genomic DNA extraction problem! - (Feb/14/2008 )

My lab is trying to do a DNA extraction from a fungus, I know it's not rocket science, but, nonetheless, the lab is having a major problem with there being no product after running the gel- infact, the only thing we do see is our ladder. Our protocol is as follows:

1. Add mycelia to a 1.5 ml tube after removing extra liquid with a kimwipe.
2. Add 100 microliters of glass beads and 500 microliters of cell lysis solution. (I had thought about using a sonicator)
3. Place on Vortex Geni II for ~20 min.
4. Spin down for 10 min at 13k rpm.
5. Add 3 microliters of RNase mix by inversion. (I'm pretty sure the RNase is DNase free.)
6. Incubate at 37 deg. C for 15 min.
7. Cool at room temperature for 5 min.
8. Add 200 microliters of protein precipitation solution.
9. Spin down for 3 min at 13-16k rpm.
10. Remove the upper phase.
11. Add 600 miroliters of isopropanol to a clean 1.5 microliter tube then the upper phase from the previous tube.
12. Mix by inversion and spin down for 1 min at 13-16k rpm.
13. Aspirate the supernatant and add 70%, room temp. ethanol.
14. Spin down for 1 min at 13-16k rpm.
15. Aspirate the ethanol and air dry the pellet
16. Add 65 microliters DNA rehydration solution and incubate for 30 minutes at 65 deg. C and 4 deg C over night.

After troubleshooting, we have found that the problem doesn't lie with the reagents because we opened up new ones; nor does the problem lie with lysing our cells because we have ran a gel with sample from previously extracted fungus from 4 years ago (we know that extraction was successful because we have the gel print outs to prove it) with our recent sample and there is still nothing to be seen, unless that older DNA is no longer active. I have run out of ideas, other than we are not loading enough sample in our gel, our current volume is 2 microliters of sample to 1 microliter of 6x dye to 3 microliter of dH20.

Any insight would be greatly appreciated.

You could try omitting the RNAse, or add EDTA to the lysis buffer to inactivate the DNAse. You can make RNAse free of DNAse by putting a closed tube in boiling water for an hour or so. Do you see a pellet? You might add pellet paint or glycogen to the supernatent prior to ethanol precipitation to make the pellet more visible. Have you tried spec measurements of DNA concentration?

What size of glass beads are you using?

It is unlikely to be the problem but from my experience on working with soil samples and bacteria (we use the Griffiths method and we combine 0.3g of glass beads and 0.2g of sand) the size of the glass beans may matter. If you check in biospec ( www.biospec.com/beads.htm ) they recommend 0.5mm diameter beads. Moreover, you may have to optimize the sample size/bead volume/extraction buffer ratio. In our soil samples we used a ratio of 1:1:1.

1. How much proteinase are you adding and how incubate it????
2. Add 2.5 vol of 100% isopropanol or absolute ethanol very cold ( I store it at -20C) and 0.5 Vol Na acetate (3M) (the DNA should be seen at this step) and incubate 1h at -80C or -20C overnight.
3. centrifuge at max rpm for at least 15 min.

Was it RNase A you were using? What's the concentration of your RNase stock? RNase A has DNase activity at high concentrations, even it has been boiled or autoclaved.

You may want to do another extraction and compare with and without RNase.

Hi,

so first of all it would be good to know which fungus you are working with! I am using several different protocols for fungal DNA to get proper PCR results.

Usually when working with fungal DNA, the reason for not working PCR are inhibitors co-extracted with DNA.

Make sure that you do not use to much mycelium in your extraction...0.5 - 1 cm2 should do the job. And be careful not to transfer any agar into your extraction! This will inhibit your PCR!
I usually 2mm glass beads work well for fungal pure cultures...
My recomendation is to try a "classical" CTAB or SDS extraction buffer, use (phenol)/cloroform/isoamylalcohol to prcipitate Proteins, Isopropanol and Ethanol to precipitate DNA and resuspend in TE or sterile water.

I do not know if you tried to use BSA in your PCR, but this usually solves a lot of problems