I have extracted genomic DNA from canola and n.tabacum using several methods, but the PCR results aren't the same. Both of the plants are recombinant. My product PCR should be 1200 bp but in PCR result there are a lot of shorter bands. The total PCR volume was20 µ L containing 2 µ L 10× Qiagen PCR buffer, 1 µ L DNA template, 1.5 mM MgCl2, 0.2 mM each dATP, dCTP,dGTP, and dTTP, 0.25 µ M each forward and reverse primer, 0.1% BSA (w/v), 1% PVP (w/v), and 0.5 U Hot-Start Taq DNA polymerase. Amplifications were carried out with thermal cyclers . The initial step of 95°C for 15min was followed by 40 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 1 min 30 s, and 1 cycle of 10 min at 72°C. PCR products were analyzed by electrophoresis on 1% agarose gels in 0.5 × TBE . Can I use less concentration of Mg? I have increased the annealing tem 1 and 2 and 3 degrees but there was no band any more.
Hello, I had the same trouble when running a PCR from microalgae using a similar PCR protocol except that I did 35 cycles and not 40, and my solution was to increase the annealing temp up to 60ºC, then only my band product of interest was in the gel and the artifacts were gone. Maybe you can try decreasing the number of cycles to 35 and to increase the annealing time to 20 seconds at 55 or higher temps.
One possibility that comes to my mind is that your recombinant plants are integrating some fragments of your plasmid or cassete in the genome and thats what you are obtaining, this has been reported for some cases when working with recombinant plants, specially if you are using integrating vectors.
Hope it helps!
Hi
As suggested above you'll need to optimize the PCR conditions carefully....
bye
As you mentioned that u have extracted DNA from different methods but u just mentioned DNA template 1µ L during reaction. so you should calibrate DNA and take same DNA conc. each time. and then try to optimize PCR conditions. such as reducing Mg as u mentioned.
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