Conditions we used were- 98deg 45s | 98deg 10s | 52deg 30s | 72deg 40s | 72deg 10mins | x35 cycles. But did not get positive results. So we tried gradient PCR to optimize the annealing temperature(48 to 59 deg).
The attached gel picture is of the gradient PCR product. Please tell me how to get crisp bands and what I can infer from these bands
Other details-
MasterMix-RedTaq (Amplicon),
Template conc.-100ng/ul for 10ul reaction
Matk forward- 5’- CGT ACA GTA CTT TTG TGT TTA CGA G- 3’
Matk reverse- 5’- ACC CAG TCC ATC TGG AAA TCT TGG TTC- 3’
Try to extend denaturations in amplification profile: 2min and 20-30s during cycles
Dear Suresh
You can use Mutation Discovery .com . It 's program working online after enter primer's gene and size band . It give you the PCR program
Are these new primers or have they been stored for a long time?
Please clarify how much template you are using...100ng/ul is 1ug in the whole reaction which may be too much and have pcr inhibitors in the dna. I would try increasing the Mg concentration a little and do a titration of dna from 10 to 100 ng total dna. If there are inhibitors then a smaller amount of dna will amplify better. Do this at 50c then if the pcr works run the gradient again
Hello
Denaturation step temperature of the reaction is too high, annealing step temperature is too low. Primers may be not specific for the DNA of plant you use for PCR.
Hi,
I suggest that you try to amplify your DNA samples with a different pair of "control" primers in order to check its quality for PCR. Maybe there is some inhibitor in your DNA samples.
Good luck,
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