I have been running a series of singleplex and multiplex PCR reactions for several weeks now using E. coli genomic DNA. I typically get anywhere between 50ng/uL and 120ng/uL for 50uL and 25uL reaction volumes respectively. I know PCR yield is dependent on several underlying factors, but I want to get a general idea of what people typically get with their vanilla 30-cycle genomic bacterial DNA PCR reactions. I want to know if the yields I am getting are typical or lower than the norm. I have tried optimizing the number of cycles and annealing temperature with little to no difference. I don't know if I have an inhibitor in my reaction or if I have reached my maximum allowable yield. Any additional comments or feedback would be greatly appreciated.
u might try increasing the concentration of the template like 200 ng in 50 microlit reaction. Also try increasing the number of cycles.
50 - 120 ng/ul sounds about right to me. 10 ul of that on an agarose gel would give you a good fat band, which is what we usually observed with a good PCR run. You can easily calculate the theoretical maximum yield: this is the concentration of the least concentrated primer (in ng/ul) x 2 x amplicon length/primer length.
Its depend upon size of your pcr product as well, Change the time of elongation 1kb/min of amplified region additional add little more polymerase enzyme upto 0.75ul. Or follow mfg protocol
Good Luck
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