I am analyzing residual DNA in samples containing a high concentration of protein, therefore I have to do the extraction protocol. However, my results have been very inconsistent, ranging from undetermined to a very large concentration in the sample (even larger than the DNA concentration I spiked the sample with). And then I noticed that ALL my samples had bubbles in the tube, while none of the standards had (photo attached). I suspect this caused my results to be out of place. Now I make sure I don't get any bubble during preparation (carefully pipetted the mix, spin it down, etc) but after running it in the PCR machine there were big bubbles in the tube containing samples. So at this point, I am thinking it may have been caused by something during the extraction process. However, I can't pinpoint what may be gone wrong. Here is what I did so far:
1. Diluting the samples to reduce the protein content
2. Longer incubation period for proteinase K
Anyone have any idea what might have caused it and what do I have to do?
Ashari Zain is possible the higher temeperature during the incubation inside the qPCR? Or using a loy of number of cycle during the qPCR?
Giorgia Pertile I use the following setup:
95 degrees Celcius for 10 min, 95 degrees Celcius for 15s, 60 degrees Celcius for 1min, for 40 cycles. I suspect that as well but it doesn't explain why it only happened to my samples and not my standards.
Ashari Zain Did you try in normal PCR with a gradient of the temperature to check if something happen?
Which protocol did you use for the DNA extraction? Is the same protocol of the standard?
Because I work with fungi and more time I see a differrent beaviuor between standard (pure culture) and DNA extracted from soil. I used the same protocol but in my case is possible that I co-extracted acid humic and this is not good for the qPCR/PCR analysis especially for te Taq polymerase.
Is possible that during your DNA extraction, you co-extracted other substance.
But how is the graph of your amplification and melting curve?
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- Analytical Chemistry
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