QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids

Table 3 A comparison of QuickStep-Cloning to other recently reported megaprimer-based cloning methods. Desirable features are highlighted in bold to facilitate comparison

Cloning method	QuickStep-Cloning	RF	ABI-REC	RAM	EMP	IFPC
Cloning strategy	Megaprimer	Megaprimer	Megaprimer	Megaprimer	Megaprimer	Megaprimer
Amplification mode	Exponential	Linear	Exponential	Exponential	Exponential	Exponential
Transformed product	Nicked-circular plasmid (2 nicks per plasmid)	Nicked-circular plasmid (2 nicks per plasmid)	Linear DNA	Linear DNA	Closed-circular plasmid	Closed-circular plasmid
E. coli cells used	Chemically competent DH5α and C41 (DE3)	Electrocompetent TG1	Chemically competent DH5α	Strain type not reported	Chemically competent DH5α	Chemically competent TOP10
In vivo homologous recombination	No	No	Yes	Yes	No	No
Enzymatic phosphorylation-ligation	No	No	No	No	Yes	Yes
Number of primers required	4	2	3	3	3	3
Gel purification	No	No	No	1×	No	Strongly recommended
PCR purification	1×	1×	No	No	2×	No
Estimated cloning time^a	5 h 15 min	14 h	7 h 45 min	7 h 45 min	7 h 15 min	6 h 30 min
Reported cloning efficiency^b	93–97 %	~90 % ^c	93–100 %	75–94 %	10–100 %	~90 %
Reference	-	[8]	[12]	[13]	[14]	[15]

^aAs estimated for cloning 1 kb DNA fragment into 7 kb plasmid according to originally reported protocol (for more information see Additional file 1)
^b Judging by the percentages reported, all methods are capable of delivering similar efficiency. Worthy of note, these numbers are dependent on the approaches used by the authors to evaluate cloning efficiency
^cAs reported in the original paper [8]. Ulrich et al. [14] and Mathieu et al. [13] demonstrate, respectively, 27 and 16 % efficiency for RF cloning

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