the attached qPCR picture shows the melting curve of my samples. the efficiency is 43.5%. I am actually targeting genomic DNA of Cryptosporidium actin gene from direct faecal extraction.
Dear Eljelani,
I guess you should discard any inhibitors in the samples, and testing serial dilutions of highly purified target DNA (or even plasmid DNA containing your target) could help. After achieving the efficiency ( and also specificity an sentitivity studies should be performed), the DNA extraction method should be analyzed. Crypstosporidium spp oocysts are difficult to broke, so I would suggest some extraction starting with physical disruption (check for example the protocol of ZR Faecal DNA kit (Zymoresearch)) or similars. In the mentioned case we know that the kit is functioning well for protozoan oocysts DNA extration, with high DNA purification (inhibitors free).
Best regards,
Gastón
Thanks More for your support, I am actually using exactly the same extraction kit for my sheep faecal materials, and it is really given great concentration and purity. I have done series dilution for my samples, but the results still the same (no improvement in the efficiency).
Dear Eljelani,
The picture you attached doesn't show the melting curve of your samples but the amplification plot, and as you say the efficiency seems to be low. I think that the problem could be due to the probe, because even you obtain amplificationin some of your samples, the florescence level obtained at the end point of your PCR reaction is quite low. May be your probe is not working well or you are not using the right concentration. Good luck.
thanks Saugar, I have prepared 10pmol probe from the stock, and then taken 0,2 micr for my qPCR. The temperature that it came with he probe is 48C, but the primer temperature 58C. I am not sure if this might affect the efficiency or not.
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