Difference between revisions of "IGEM:Peking/2007/Switch: PCR"
|Line 48:||Line 48:|
4. 72℃ ETs
4. 72℃ ETs /min
5. 72℃ 10min
5. 72℃ 10min
Revision as of 08:22, 18 October 2007
Polymerase Chain Reaction
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.
一般Taq系列聚合酶的体系为模板1ng~1ug（取决于靶序列含量），dNTP 200uM每种，引物50pmol每种，酶1U，缓冲液和水。模板可以是质粒，Lambda DNA，菌落和基因组。高特异性引物以20bp为宜，低特异性引物可长至30~35bp。
Taq series polymerase reaction components
10×PCR buffer（Ex/LA） 5uL
Ex/LA Taq 0.25uL
10×PCR buffer（Ex/LA） 1.5uL
Ex/LA Taq 0.1uL
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