Polymerase Chain Reaction ( PCR ) for gDNA

Usage amount per sample:

Material ^※	Concentration	Addition per sample	Location
gDNA ^†	10 ng / uL	4 μL	Usually -20°C
F-primer		0.4 μL	-20°C ( 2-3-4 )
R-primer		0.4 μL	-20°C ( 2-3-4 )
2X-Taq (orange lid)		5 μL	-20°C ( 4-3-4 )
Ambion water		0.2 μL	RNA bench
Total volume:		10 μL	place on ice

^※ All materials should be placed on ice.

^† gDNA : The gDNA stock solution of all samples should be standardized to the same concentration, for example 10 ng/μL.

Procedure

Prepare the total amount of the required working solution ( F-primer, R-primer, 2X-Taq );

For example, if we had 10 samples, then me make the working solution by adding 4 μL F-primer, 4 μL R-primer, and 50 μL 2X-Taq into a new 200 μL PCR tube.

Dispense the working solution into a new 200 μL PCR tubes ( 5.8 μL for each sample );
Lastly, add the sample gDNA stock solution;
Close the lid tightly, and swipe the tube gently with finger several time to mix the solution well;
Slightly centrifuge to bring down the solution;
Put into the PCR machine ( the machine should be warmed up around 2 mins before use ).

Process	Temperature (°C)	Time	Cycle
Initial denaturation	95	1 min	--
Denaturation	95	15 secs	30
Annealing ^†	56	15 secs	30
Extension	72	1 min	30
Complete extension	72	5 min	--
PCR product storage	4	∞	--

^† Annealing temperature depends on primers.

If you encounter any issues with the protocol, please report them at:
https://github.com/P10911004-NPUST/jklai/issues.