推荐：PCR与RT-PCR技术--生物秀

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。

平端PCR克隆方法（Zero Blunt PCR Cloning Kit）

Zero Blunt PCR克隆乃利用传统T4连接酶方法，但载体和Topo平端的一样，含ccdB基因，所以同样可降低菌落背景，易于筛选。

表10 PCR克隆试剂盒的选择

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	K4500-01 K4550-01 K4560-01
				•Blue/white color screening
			TOPO TA Cloning® Kit Dual Promoter	•≥95% recombinants	K4600-01 K4650-01 K4660-01
				•Blue/white color screening
				•Dual Sp6 and T7 promoter primer sites for in vitro transcription
			TOPO TA Cloning® Kit for Sequencing	•≥95% recombinants	K4575-01
				•Streamlined sequence analysis
		Ligase-mediated(overnight)T4 DNA ligase	Original TA Cloning® Kit	•≥80% recombinants	K2000-10 K2030-10
				•Blue/white color screening
			Original TA Cloning® Kit Dual Promoter	•≥80% recombinants	K2050-01 K2060-01
				•Blue/white color screening
				•Dual Sp6 and T7 promoter/primers sites for in vitro transcription
		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	K2800-20
				•Positive selection resulting in low background
			Zero Blunt® TOPO® PCR Cloning Kit for Sequencing	•≥95% recombinants	K2875-20
				•Positive selection resulting in low background
				•Streamlined sequence analysis
		Ligase-mediated(1 hour)T4 DNA ligase	Zero Blunt® PCR Cloning Kit	•≥95% recombinants	K2700-20
				•Positive selection resulting in low background
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	K4700-10
				•Positive selection resulting in low background

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