A ligation and restriction enzyme independent cloning technique: an alternative to conventional methods for cloning hard-to-clone gene segments in the influenza reverse genetics system

Table 3 Comparison of relative time taken by LREI cloning compared to conventional cloning

Steps in cloning procedure		Time required during different cloning steps
Steps in cloning procedure		Conventional cloning	Ligation and Restriction Enzyme Independent (LREI) Cloning^a
a	Viral RNA extraction and cDNA synthesis	2.5 h	2.5 h
b	Generation of desired amplicon by thermocycling	3–5 h	3–5 h
c	Agarose gel electrophoresis and gel extraction of desired amplicon	2 h	2 h
d	Restriction digestion of desired amplicon and Restriction digestion of the cloning vector	1 h – 16 h (depending upon the enzyme used)	Not required
e	Purification and quantification of the digested amplicon and the cloning vector	1–2 h
f	Quick ligation or overnight ligation of the digested amplicon and cloning vector	1 h – 16 h (depending upon the ligation kit)
g	Transformation	1.5 h	1.5 h
h	Screening of positive colonies	3–7 h	3–7 h

^aOur LREI cloning saves 2 days of time while cloning and is more efficient in cloning unstable polymerase genes into standard cloning vector

ISSN: 1743-422X