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. 以上3种Taq酶都可以在产物末端加A,因此它们的PCR产物可以直接连接T载体。

5. KOD plus的延伸速度,保真度均比Taq酶有明显提高。但是由于其3’->5’外切活性,KOD的扩增能力低于Taq系列的聚合酶。克隆基因应当首选KOD plus,只有KOD plus无法得到有效扩增时才考虑其他的聚合酶。

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

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