IGEM:Peking/2007/Switch: PCR: Difference between revisions
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== | ==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 | Template 1uL | ||
| Line 31: | Line 33: | ||
50uL system | 50uL system | ||
=== | ====colony PCR==== | ||
10×PCR buffer（Ex/LA） 1.5uL | 10×PCR buffer（Ex/LA） 1.5uL | ||
| Line 46: | Line 48: | ||
15uL system | 15uL system | ||
=== | ===Taq series polymerase reaction condition=== | ||
1．94℃ 5min | 1．94℃ 5min Taq enzyme activation by heat | ||
2．94℃ 30s | 2．94℃ 30s DNA denaturing | ||
3．Tm-5~10℃ 30s | 3．Tm-5~10℃ 30s Tm is annealing temperature, with a range of 45～68℃ | ||
4. 72℃ ETs | 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 | 5. 72℃ 10min add A at the end of each fragment | ||
Latest revision as of 08:25, 18 October 2007
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
