Let's say if we purchased ready master mix from certain manufacturer and the instruction says that Ta is based on Tm of primer (my F primer Tm is 59 and R primer Tm is 62), then how we can set the Ta? and if PCR product produced faint band, do we need to optimize the Ta? When should we optimise it and how?
I have instinct,that the master mix is engineered to make such a way that we dont need to go for temperature gradient and identify the correct Ta. Am i right?
I attached the instruction to set up the PCR reaction.
If you want to save some master mix (and money), you can do it by the "3 rules":
First run, three conditions:
1. you try the Ta recommended by the master mix insert (f.e. for Roche master mixes often "Tm of primers -5°C")
2. 5°C below the first temperature
3. 5°c above the first temperature.
=> you can see if you need to increase the temperature, go down or keep the advised temperature. (Some time even, it doesn't matter)
If you don't have perfect results at first run, you can try again 3 temperatures in the direction furnished by the first experiment (lower or higher temperature)
Thanks so much for your help, really appreciate it. I will try identifying the best Ta for my amplified gene of interest and see the results tomorrow. I never did PCR during undergrad years and this was the first time ever.
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