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

From: 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

Steps in cloning procedureTime required during different cloning steps
Conventional cloningLigation and Restriction Enzyme Independent (LREI) Cloninga
aViral RNA extraction and cDNA synthesis2.5 h2.5 h
bGeneration of desired amplicon by thermocycling3–5 h3–5 h
cAgarose gel electrophoresis and gel extraction of desired amplicon2 h2 h
dRestriction digestion of desired amplicon and Restriction digestion of the cloning vector1 h – 16 h (depending upon the enzyme used)Not required
ePurification and quantification of the digested amplicon and the cloning vector1–2 h
fQuick ligation or overnight ligation of the digested amplicon and cloning vector1 h – 16 h (depending upon the ligation kit)
gTransformation1.5 h1.5 h
hScreening of positive colonies3–7 h3–7 h
  1. aOur LREI cloning saves 2 days of time while cloning and is more efficient in cloning unstable polymerase genes into standard cloning vector