Greetings. I’m currently running a nested pcr for giardia. My mastermix comprised of 3mM Mgcl2, 5unit of taq polymerase, 0.2uM of forward and reverse primers each respectively and 0.2mM of dNtp and 2ul of dna in a total of 25ul pcr reaction. The annealing temperature is 58 degree celcous for 35 cycles of PCR. My target base pair is around 530bp. I attached the gel picture, the one with band is my positive control. Thank you for any guidance and help.
Hello, it seems like the template DNA used in the PCR had high concentration which might inhibit the PCR amplification. You did not mention the concentration of the template DNA (only volume mention). Check it first, if it is in the range of template concentration. If it is too high, dilute it accordingly.
Set up the PCR with higher annealing temperature to avoid non-specific binding, if needed. Also, reduction of extension time can help further to omit non-specific amplification of higher molecular size.
All the best!
5u of taq is far too much . 0.5u is more reasonable. You have to be very careful about over amplification of the second nested pcr. Typically use a 1:1000 dilution of the first round product and run 20-25 cycles of the second round pcr to get clean amplimers. I think that you problem is over amplification
Satyendra Mondal my 2 DNA samples concentration are 34.75 ng/ul and 24.6 ng/ul respectively.
My current nested pcr cycle are 94 degree celcius for 5mins (initial denaturation), 94 degree celcius for 45 seconds (denaturation) 50 degree celcius for 45 seconds (58 for 45 seconds in secondary pcr) (annealing), 72 degree for 1 minute (extension) and final extension (72 degree celcius for 10 min) X 35 cycles (for both pcr)
Thank you so much for your help
Paul Rutland i really appreciate the suggestion. i will try to run the pcr according to it and update the result soon. Thank you!
Hi dear.
It seems that you over loaded the master mix in the well. Dilute it.
- Badges
- Science topic
- Similar topics
- Filing