bjk1985

Maybe a dumb Q... but I've been doing a protocol for DNA isolation from soil that has a couple of precipitation steps that, after addition of solution and brief vortexing, says to incubate at 4 C for 5 min. I've just been sticking them in ice (a bucket of ice that itself is at RT). Can you see any problems in doing this?

Tried with the Promega Wizard kit, has worked fine for plant tissue and bacterial cells from past experience, but not mycelia (in all cases material was first ground using mortar/pestle & liquid nitrogen). Then I tried something I thought should work (using proteinase K) but that was even worse for getting DNA. My first step was to use RNase A at 37 C in buffer of 150 mM tris, 25 mM EDTA, and 1% SDS, but that didn't seem to work at all (and this was before proK). Nanodrop data for all was quite good in terms of nucleic acid purity; on the gel I loaded 200-250 ng of each sample.

I don't know why I am getting such terrible results, both ways. Anyone get good DNA from fungus & how do you do it? I'm working with the Diaporthe sp. and Phomopsis sp., btw, which are both Ascomycota.

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A soybean pathogen in the Ascomycota, no other DNA present (at least not intentionally). Amplicon in the ITS region of rDNA which as i understand has many copies per genome. First one is 20-25 ng, and then each of the next four are diluted tenfold from the previous. Based on what I've read and what another student did with these same materials, I think for these amounts that the Ct values on this should start to come in much sooner (like in the 10-15 range).

First one has 0.1 mM Tris (rxns are 20 uL), and the other 4 have bascially 0.05 mM. From what I've looked up as well as experience with standard PCR, this amount should not have any effect. Primers were 200 nM and probe was 100 nM (both working stocks were 10 uM and in 10:1 mM TE). Original DNA was in 10:1 mM TE, and then each dilution done in 10 mM Tris (amount of each that was added was 2 uL).

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The other student used Taqman Universal MM, and I used Taqman Genotyping MM (tech support said it wouldn't matter). I did just receive the former, though, & will be trying that next time. Anyone have any thoughts/experience on this assuming that it's not the MM? The exponential phase does seem a little flat, maybe inhibitors... the earlier ones seem to be worse, or am I reading too much/incorrectly into that?

The intention, btw, is to detect and quantify the pathogen in soil and plant tissue samples, and I fear these high Ct values will be hard to separate from potential "background" amplification that seems to be coming in the mid-30s (although this needs to be assayed to see whether it's contamination from the target DNA, or if it's something else).


Note: The DNA used was the PL*. It's actually a bit more (~230 ng) than the wheat (~160 ng), but for me fungi have been a pain to get high-quality gDNA (compared to plants and bacteria), and I have to now always gotten more faint bands (relative to the wheat) when loading similar amounts based on the A260.

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