My PCR Protocol was:
Hot start: 95 °C for 10 min
Denaturation: 15 s at 94°C,
Annealing for 30 s at 61°C,
Extension step for 30 s at 72°C;
Final extension for 10 min at 72°C;
A hold at 6°C.
For 35 cycles
Ahlam Issam Mujahed
Usually when you got a non specific bands, you need to play with the annealing temp. and time. Also you can play with MgCl2 concentration you added in the reaction.
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Vijaya R. Chitnis
You can determine optimum annealing temperature my running gradient PCR, in addition you can reduce final extension time 2-5 min would be better.
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