Table 1.

Optimization of multi-fragment ligation conditions

	Inserts : Plasmid Backbone Ratio	Number of Colonies	Transformation efficiency (cfu/μg)	Positive clones (%) (N=4)
PHLEO	0.5:1	450	3.2 × 106	25
	1:1	900	6.4 × 106	75
	2:1	1180	8.4 × 106	75
	3:1	1500	1.1 × 107	75
PAC	0.5:1	524	3.7 × 106	25
	1:1	1110	7.9 × 106	75
	2:1	1467	1.0 × 107	100
	3:1	1880	1.3 × 107	75
pUC19	-	2000	4.8 × 108	-
Plasmid backbone fragment	-	70	5.0 × 105	-

The molar ratio of inserts (5’- and 3’- IMPDH TS fragments and the drug resistance cassette) to plasmid backbone was varied with respect to 10 ng of the plasmid backbone. The Inserts: Plasmid Backbone Ratio denotes the molar ratio of each insert fragment relative to the plasmid backbone. The number of colonies from plating 100 μl (21% of total transformation volume) of E.coli transformed with 2 μl ligation reaction (representing 0.67 ng plasmid backbone fragment) is presented. Transformation efficiencies are given as colony forming units per μg of DNA and are calculated based on 0.67 ng of plasmid backbone fragment per transformation. Plasmid pUC19 (0.02 ng) was included to assess the transformation efficiency of the lot of commercially prepared competent E. coli used in this experiment. Transformation of a ligation reaction performed using only 10 ng of the purified plasmid backbone fragment (originating from the same batch used in inserts:vector ratio optimization) served to indicate the contribution of pBB-GFP to background. Further assessment of transformants derived from ligation reactions using 2:1 inserts to plasmid backbone ratio showed that the percentage of clones positive for the correct PHLEO- or PAC-containing targeting vector was 75% and 85%, respectively (N=20).