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Good standard curve (maybe) but Ct values high


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#1 bjk1985

bjk1985

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Posted 08 February 2013 - 10:14 PM

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).

PCR curve.bmp.jpg

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.

Picture 001.jpg

Edited by bjk1985, 08 February 2013 - 10:32 PM.





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