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Topo平端克隆方法和长PCR片断克隆法（Zero Blunt Topo PCR Cloning Kit）（Topo XL PCR Cloning Kit） 

    因为越来越多高确限度热稳定酶如Pfx、Pfu被用于PCR扩增，使到PCR后都只是平端产物。这种平端克隆往往效率低，并且有非特异性背景菌落产生，造成筛选困难。 
    Topo平端克隆主要改良载体设计，把ccdB （Control of cell death）基因并入到mcs中，如果没有DNA片断插入，ccdB基因则会表达，ccdB基因的表达蛋白，会破坏细菌的DNA gyrase，造成细菌染色体的降解导致细菌死亡。如果有插入片断，则ccdB基因表达受到阻碍，所以细胞可生长。这样可以把高背景的缺点改善，节省筛选的时间。Topo平端克隆与Topo TA方法一样，操作简单快速。 

TOPO PCR表达克隆法 
   
    传统限制酶切法必须把目标片段切下来再连接至表达载体上，这会导致一些非原始蛋白的其他氨基酸也混在其中，从而影响蛋白表达和蛋白的功能。利用TOPO克隆技术把PCR产物直接克隆至表达载体，则可避免这种情况。 

  TOPO PCR表达克隆法仍利用特异引物把目标基因PCR扩增后，再利用高效的Topoisomerase 酶把产物快速连接到T表达载体上，这是到载体上的目标基因不含非编码区，Invitrogen提供了原核细胞、酵母、昆虫细胞和哺乳动物细胞的各种PCR克隆表达系统。
　

Amplification Enzyme

Application of Cloned PCR Product

Ligation Method

Product

Advantages

Cat. No.

Taq DNA Polymerase

•Sequencing
•Safeguarding sample
•In vitro transcription

TOPO® Cloning (5 minutes) Topoisomerase

TOPO TA Cloning® Kit

•≥95% recombinants
•Blue/white color screening

K4500-01
K4550-01
K4560-01

TOPO TA Cloning® Kit Dual Promoter

•≥95% recombinants
•Blue/white color screening
•Dual Sp6 and T7 promoter primer sites for in vitro transcription

K4600-01
K4650-01
K4660-01

TOPO TA Cloning® Kit for Sequencing

•≥95% recombinants
•Streamlined sequence analysis

K4575-01

Ligase-mediated(overnight)T4 DNA ligase

Original TA Cloning® Kit

•≥80% recombinants
•Blue/white color screening

K2000-01
K2030-01

Original TA Cloning® Kit Dual Promoter

•≥80% recombinants
•Blue/white color screening
•Dual Sp6 and T7 promoter/primers sites for in vitro transcription

K2050-01
K2060-01

TOPO® Cloning(5 minutes) Topoisomerase

Various

•Fast cloning directly into an expression vector

All the TOPO Exp. Kits

Proofreading polymerase
高确限度酶

•Expression of cloned PCR product

TOPO® Cloning(5 minutes) Topoisomerase

Zero Blunt® TOPO® PCR Cloning Kit

•≥95% recombinants
•Positive selection resulting in low background

K2800-20

Zero Blunt® TOPO® PCR Cloning Kit for Sequencing

•≥95% recombinants
•Positive selection resulting in low background
•Streamlined sequence analysis

K2875-20

Ligase-mediated(1 hour)T4 DNA ligase

Zero Blunt® PCR Cloning Kit

•≥95% recombinants
•Positive selection resulting in low background

K2700-20

Polymerase mixturesfor long Template混合酶

•Sequencing
•Safeguarding sample
•In vitro transcription

TOPO® Cloning(5 minutes) Topoisomerase

TOPO® XL PCR Cloning Kit

•High-efficiency cloning of long (3-10kb) PCR products
•Positive selection resulting in low background

K4700-10

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