Hi,
I am looking for advice. I designed my own primers for human TBP, TGFB1 and ITGB6 Gene's. To test my primers I ran a PCR and gel, however, it didn't work.
Does anyone have any solutions? I am thinking of trying an annealing gradient PCR to check primer efficacy?
1) You can try doing gradient pcr to find out the best annealing temp.
2) There are smearing at the top of the gel, so you may need to reduce the concentration of the dna that you are using, dna concetration should not be more than 250ng/50ul reaction.
@Vikki Wong
Thank you, I will try a gradient PCR.
The DNA extracted was at 38.45ng/ul. 1.25ul was used in a 25ul reaction.
I used 12.5ul promega PCR master mix (2x conc) 2ul template DNA, 1.25 primers each of forward and reverse, 1.25 BSA and 6.75ul of pcr water
Hi Lucy Butler
It often happens that the primers are no working properly. Try to find a suitable annealing temperature as per Wong Ling Chie suggestions. You can also try with different enzyme premix to check your PCR products.
Thanks
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