IGEM:Peking/2007/Switch: PCR

Choose enzymes
1.Taq has no 3'->5'exonuclease activity, Taq has a low fidelity. Taq can only be used in testing but not in cloning genes.

2.Ex Taq and LA Taq are modified Taq by Takara, they have 3’->5’exonuclease activity and a relatively high fidelity. 3.Generally speaking, genes shorter than 800bp can be cloned by Ex Taq. Genes shorter than 1.5kb can be cloned by LA Taq. Ex Taq has the highest amplification efficiency.

4.Taq can add A at the end of each fragment, so their PCR product can direcly link to T plasmid.

5.KOD plus has a higher fidelity than Taq. KOD has 3’->5’exonuclease activity and thus lower ability than Taq series enzyme. Cloning a gene should first choose KOD plus.

PCR system
Normal Taq polymerase reaction component has a template of 1ng~1ug, dNTP 200uM each，primer 50pmol each，polymerase 1U，buffer and water. Template can be plasmid, Lambda DNA，colony and genomic DNA.

Cloning
Template		1uL

10×PCR buffer（Ex/LA）	5uL

10×dNTP			5uL

Primer-F		1uL

Primer-R		1uL

Ex/LA Taq		0.25uL

ddH2O			36.75uL

50uL system

colony PCR
10×PCR buffer（Ex/LA）	1.5uL

10×dNTP			1.5uL

Primer-F		0.5uL

Primer-R		0.5uL

Ex/LA Taq		0.1uL

ddH2O			11uL

15uL system

Taq series polymerase reaction condition
1．94℃ 5min		Taq enzyme activation by heat

2．94℃ 30s		DNA denaturing

3．Tm-5~10℃ 30s	Tm is annealing temperature, with a range of 45～68℃

4. 72℃ ETs		ET is elongation time，1kb/min

25 cycles，more cycles can produce more products but also introduce more mutations

5. 72℃ 10min		add A at the end of each fragment