IGEM:Peking/2007/Switch: PCR: Difference between revisions
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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. | 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. | 4.Taq can add A at the end of each fragment, so their PCR product can direcly link to T plasmid. | ||
5. | 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体系=== | ===PCR体系=== |
Revision as of 08:19, 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体系
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。
常用的Taq系列聚合酶反应体系
克隆基因
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
菌落PCR检测
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
一般Taq系列聚合酶的反应条件
1.94℃ 5min Taq酶热激活
2.94℃ 30s DNA变性
3.Tm-5~10℃ 30s Tm为引物退火温度,范围45~68℃,以60~68℃为宜
4. 72℃ ETs ET为延伸时间,1kb/min
重复2~4,25个循环,增加循环数可以增大产物量,但是会引入更多突变。
5. 72℃ 10min 在产物末端加A,不需要加A时可省去此步