For fungal identification I will be using TEF ALPHA tricho primer which has Tm 52°C. I wanted to know the PCR conditions as even by setting the annealing temperature at 48°C I was getting multiple bands and at 56°C I got no bands (image attached). Please guide for PCR condition for TEF ALPHA tricho primer.
A good strategy to determine an optimum annealing temperature is by using Gradient PCR. You can also find similar discussion here:
https://www.researchgate.net/post/How-should-one-perform-gradient-PCR-and-how-much-time-will-it-take
Shortly, in gradient PCR, normally 6-8 PCR reactions are set up in different tubes, and then these tubes are subject to PCR in a single run. In the PCR program, the annealing temperature is set ranging from "Tm-6 to Tm-1". In your case, you can set the annealing temperatures from 48°C to 55°C. After the completion of PCR, you can find on agarose gel electrophoresis, which annealing temperature is giving a single band.
Looking at the non-separation of your marker (DNA ladder), I think you may be dealing with some issues with your running buffer. It seemed more like smearing, rather than annealing temperature failure
The conditions will depend heavily on the Taq or mastermix you are using. They usually come with a manual that will give a range for the cycling conditions.
Use those as your base protocol, but run a gradient for the annealing temperature of up to 4 degrees above and below the current temperature.
I do agree with Bashiru Garba that your gel doesn't have good separation. What percentage gel is this and what voltage/time/mA did you run it at?
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