Thứ Tư, 15 tháng 5, 2019

PCR amplification not working?

By the way in the PCR reaction include:
DNA template (pure or include inhibition compounds)
Primer
Master mix (include dNTP, DNA polymerase, buffer - it could be optimized)

The reaction happen in thermometer:
Denaturing - when the double-stranded template DNA is heated to separate it into two single strands.

Annealing - when the temperature is lowered to enable the DNA primers to attach to the template DNA.

Extending - when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme

notice: 
Denaturing depend on enzyme - it could be 94oC eg. Dreamtaq or 98oC eg. Phusion
Annealing - it depend on primer to decide the opt temperature of annealing - low temperature easy to hold primer with DNA but less specific - high temperature need more link between primer and DNA to hold so it more specific. (from it possibly from 48oC to 64oC depend on each primer and ration AT-GC, more GC should be higher temp- was calculating by online tool)
Extending - 72oC for 1-2min depend on speech reaction of enzyme (1kb/min or 1,5kb/min) and the length of product (500bp or 1kbp) to decide the opt time to extend.

- the temperature to optimize- form low to high - to define the high temp give the acurrency result.
- the amount of DNA template (less or more) define easy or difficult to annealing the pcr products (use raw extract DNA in phenol-chloroform or use purified DNA)
- the component of reaction should be control and avoid any inhibition to PCR.





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