IGEM:Peking/2007/Switch: PCR

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Contents

Polymerase Chain Reaction

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.

Taq series polymerase reaction components

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

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