I extracted my DNA from Listeria monocytogenes pure colonies and tried to adjust the conditions of my RAPD-PCR with three primers:
HLWL74 (TA = 29.6 ºC)
HLWL85 (TA = 30.2 ºC)
OMP-01 (TA = 35.3 ºC)
I prepared separated reactions with 400 uM dNTP, 0.4 uM primer, 3 mM MgCl2, 1 X buffer, 1U polymerase, and 50 - 100 ng DNA in a final volume of 25 ul.
My PCR conditions were:
1 cycle of 94 ºC for 5 min
35 cycles of (94 ºC for 45 s, 36 ºC for 1 min, and 72 ºC for 2 min)
A final cycle of 72 ºC for 5 min.
With these conditions, I obtained bands (not the best result, you can see there is some smearing and different intensity bands, which make it difficult to identify each sample) only for primer OMP-01 and only one time. I keep repeating with the same setup but cannot get any result.
I loaded the 1,8 % agarose gel with 6 ul of PCR product and leave it for 7 hours at 30 V.
With annealing temperatures of 29-35 you are optimistic to think that they will all work at 36c annealing. Drop the Ta to 27 and when everything works raise the annealing temperature in small steps to get the best results. If you have a gradient pcr machine run a gradient pf 25-36 c. Your primers are not all annealing
Check the quality and quantity of DNA and run gradient PCR. Hope this may work for you.
yes you have to low the annealing temperature and not only used the 10 mere primers but also use 20, it will give you more match.
I performed a gradient PCR as Paul Rutland suggested, but it seems that annealing temperature is not the problem (I tried gradient from 30 ºC to 40 ºC). OMP-01 primer works very well in all the range, but neither HLWL74 nor HLWL85 (not shown) gives any amplification.
I also don't know why the big overlapped band appears in the gel (marked in the image), is it some electrophoresis trouble?
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