I have extracted good quality DNA (1339.3)ng/ul. I first run the normal PCR using same primer and i obtained very good result, but when qPCR was run , the result was totally disappointing. I used the following protocol for the qPCR;
2mnt at 50oC, 5 mnt at 95oC, 40 cycles of 20s at 94oC and 30s at annealing temp. and 1 mnt at 72oC for signal collection.
1 ul of 1/10 DNA dilution was used. and o.5 ul of each forward and reversed primer.
Thank you Dr. Florian for your response.
Actually I'm using Bacterial DNA and I used SYBR green not probe. And the PCR cycler is PIKOREA 96 real time PCR system. The amplicon size was 320bp and was not CG-rich. As it contained only about 14% of both the C and G
Thank you @ Dr. Florian
And please do you have any idea why is it impt to start with temp ( 52oC) during the initial qPCR cycle.
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