IGEM:Peking/2007/Switch: PCR

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Current revision (11:25, 18 October 2007) (view source)
(PCR system)
 
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==聚合酶链式反应==
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==Polymerase Chain Reaction==
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===酶的选取===
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===Choose enzymes===
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  Taq酶不具有3’->5’外切活性,保真度低,一般只作检测用,不用来克隆基因。
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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.
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    Ex Taq与LA Taq是Takara改造后的Taq酶,具有部分3’->5’外切活性,保真度有所提高。LA Taq更适用于PCR长链和GC含量高的模板。一般800bp以下基因可以考虑用Ex Taq进行克隆。1.5kb以下基因可以考虑用LA Taq进行克隆。Ex Taq的扩增效率最高。
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2.Ex Taq and LA Taq are modified Taq by Takara, they have 3’->5’exonuclease activity and a relatively high fidelity.
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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.
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    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。
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4.Taq can add A at the end of each fragment, so their PCR product can direcly link to T plasmid.
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    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。
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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.
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===PCR体系===
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===PCR system===
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    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。
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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.
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===常用的Taq系列聚合酶反应体系===
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===Taq series polymerase reaction components===
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克隆基因用
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====Cloning====
Template 1uL
Template 1uL
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50uL system
50uL system
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===菌落PCR检测用===
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====colony PCR====
10×PCR buffer(Ex/LA) 1.5uL
10×PCR buffer(Ex/LA) 1.5uL
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15uL system
15uL system
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===一般Taq系列聚合酶的反应条件为===
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===Taq series polymerase reaction condition===
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1.94℃ 5min Taq酶热激活
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1.94℃ 5min Taq enzyme activation by heat
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2.94℃ 30s DNA变性
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2.94℃ 30s DNA denaturing
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3.Tm-5~10℃ 30s Tm为引物退火温度,范围45~68℃,以60~68℃为宜
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3.Tm-5~10℃ 30s Tm is annealing temperature, with a range of 45~68℃
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4. 72℃ ETs ET为延伸时间,1kb/min
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4. 72℃ ETs ET is elongation time,1kb/min
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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。
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25 cycles,more cycles can produce more products but also introduce more mutations
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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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5. 72℃ 10min add A at the end of each fragment

Current revision

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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