I am running a Nested PCR on blood DNA. Although the primers have worked successfully in previous publications, I am not getting a clear bands for sequencing. The first PCR reaction mix containing 20 µg of BSA, 5% of DMSO, 1.6 mM of MgCl2, 0.5 of each dNTP, 0.7 µM of primers, 2 U of Taq polimerase, and 200 to 500 ng of the extracted DNA. The second PCR reaction was carried containing 10 µg of BSA, 5% of DMSO, 4 mM of MgCl2, 0.7 mM of each dNTP, 0.3 µM of primers, 1.5 U of Taq polymerase, and 1 uL of the PCR1 product.
The first thermal profile consisted of 95 °C for 3 min, followed by 40 cycles at 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min. And final extension step of 7 min at 72 °C. The second thermal profile was 3 min at 95 °C followed by 16 cycles of a touchdown protocol at 94 °C for 40 s, decreasing the annealing temperature from 60 °C to 45 °C for 40 s (1 °C/cycle), followed by 72 °C for 1 min. Then, 30 cycles at 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min, with a final extension step of 7 min at 72 °C.
I have followed the published methods, but I am still not successful. Could someone provide insights to improve my reaction?
Thank you in advance
i think that you have massive overamplification and the pcr product is annealing to other product molecules quite randomly and producing these smears. 30 cycles of pcr could be an amplification of 1,000,000,000 times.
Also 1ul of pcr product contains a lot of first round primer to react with the nested primers.
Try diluting the first round product 1:100 and use 1ul of this. Your cycle number should not be more than 25 and if possible I would set up 6 identical nested pcr reactions and remove 1 tube at 14, 16, 18 ,20,22 etc cycles to see how many cycles give good product in good amounts
Dear Paul Rutland.
Thank you so much for your answer. Following your suggestion, I tested the influence of cycle numbers, and performed a serial dilution of the PCR1 product (1:1 to 1:1000).
I am happy to report that it worked!! I got a nice product at 14 cycles and dilutions ranging from 1:250 to 1:750.
Cheers
Thank you Angela. It is very helpful to get feedback about what works well and I am delighted that you are getting better results.
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