Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V

BMC Biotechnology

Table 2 Cloning efficiencies using a proofreading DNA polymerase

Ligation reaction	EndoV	Number of colonies	Fraction positive
pBSK + Mitf PCR product	+	997	97.5% (39/40)
pBSK + Mitf PCR product	-	5	ND
Mitf PCR product	+	0
pBSK		10	ND
pIRES2-EGFP + AmpR PCR product	+	384	100%
pIRES2-EGFP		0
pUC19 + mRFP1 PCR product	+	113	85.8%
pUC19 + mRFP1 PCR product	-	26	100%
mRFP1 PCR product	+	0
pUC19		0

Ligation reactions were set up using plasmid vector DNA linearized by restriction endonuclease treatment. Column two indicates whether the PCR-generated insert was treated with endonuclease V. pBSK denotes pBluescript II KS(+). Fractions of positive clones were determined by red fluorescence (mRFP1 PCR product), growth in the presence of ampicillin (AmpR PCR product) or colony PCR (Mitf PCR product, 40 clones tested), respectively. ND = not determined.

ISSN: 1472-6750