There are three kinds of DNA polymerases commonly used in the laboratory : TaKaRa Taq^TM，TaKaRa E_XTaq^TM and Pyrobest^TM DNA Polymerase.TaKaRa Taq^TM is a common DNA polymerase with poor fidelity but low price, which is generally used for gene expression detection, etc. TaKaRa E_XTaq^TM is a thermostable DNA polymerase with Proof reading activity, which has certain fidelity. Moreover, the PCR product 3 'end amplified by TaKaRa E_XTaq^TM is attached with a "A" base. If you want to clone the product directly to T-vector, you can use this enzyme.Pyrobest^TM DNA polymerase is also a thermostable DNA polymerase with Proof reading activity, which is characterized by high fidelity and the amplified PCR product is a smooth terminal. Use this enzyme if you want to amplify the gene.

1.The reaction fluid is modulated in the PCR reaction tube according to the following composition:

Formulation of TaKaRa Taq^TM or TaKaRa E_XTaq^TM

Reagent	Quantity, for 50µl of reaction mixture
10X PCR buffer (MgCl2+ free)	5µl
MgCl2 （25mM）	such as TaKaRa TaqTM add 3µl
	such as TaKaRa EXTaqTM add 4µl
2.5mM dNTP mix	4µl
10µl Primer upper reaches	1µl
10µl Primer lower reaches	1µl
Template DNA	1µl
Taq or EXTaqDNA Polymerase	0.25µl
Sterile deionized water	Up to 50µl
Total	50µl//Sample

Formulation of Pyrobest^TM DNA Polymerase

Reagent	Quantity, for 50µl of reaction mixture
10X Pyrobest buffer	5µl
2.5mM dNTP mix	4µl
10µl Primer upper reaches	1µl
10µl Primer lower reaches	1µl
PyrobestTM DNA Polymerase	0.25µl
Sterile deionized water	Up to 50µl
Total	50µl//Sample

①. The total volume of the reaction is regulated according to the actual situation, and 20-50 μ l can be made to save reagents;

②.Add the components in the above table to 0.2ml or 0.5ml sterilized PCR thin-walled tube;

③. If the heating cover of PCR instrument is not used, add 30-50 μ l mineral oil to the upper layer of reaction mixture to prevent the sample was evaporated during PCR;

2.Carry out PCR amplification according to the following procedure.

PCR reaction conditions are different depending on the structural conditions of template and primer. In practical operation, it needs to be optimized according to the specific situation and PCR results.

Step	Temperature, °C	Time, min	Number of cycles	Note
Initial denaturation	94~95	1-3	1
Denaturation	94~95	0.5-2	25-35	The annealing temperature is about 5 ° C lower than the theoretical annealing temperature, and then optimized according to the reaction results.
Annealing	37-70	0.5-2
Extend	70-75	Depending on the size of the amplification product		1000 bp per minute
Final extension	70-75	10	1

At the end of the reaction, 5 μl of the amplified sample was taken, and the amplification results were analyzed by agarose gel electrophoresis. The size of the amplified fragment was determined by DNA marker or it was cryopreserved for later analysis.