Table 2.

Efficiency of the MSS MR recombination scheme

Step 1							Step 2
Construct (∼14.5 kb)	Target	Size (kb)	# colonies screened	% FLb	% WT	% Mut	Construct (∼28.5 kb)	Target	Size (kb)	# colonies screened	% FLb	% WT	% Mut
WT-Ca	BAC	163.7	50	4	100	NA	NA	NA	NA	NA	NA	NA	NA
M1	BAC	163.7	111	4	0	100	M1-L	BAC	163.7	89	38	17	83
M2	WT-Pa	∼49	18	11	45	55	M2-L	WT-Pa	∼49	9	89	44	56
M3	WT-Pa	∼49	20	20	0	100	M3-L	WT-Pa	∼49	18	100	44	56

The MSSMR reactions were performed using either BAC DNA (for isolation of DHC64C^wt and M1 clones) or DHC64C^wt in P[acman] (for M2 and M3) as the target. Using the smaller target was 2–5x more efficient than using the BAC target, but mutants were isolated at each step regardless of the target used.

^aThe wild-type recombineering construct (WT-C) consisted of the M1 construct double cut with MluI and NotI to linearize, thereby removing the “M” segment. This reaction required only one recombineering step to obtain the full length product; the designation NA (not applicable) is therefore used for Step 1, % Mutant and all Step 2 fields. This experiment was done only as a control for comparison of the efficiency of one-step vs. two-step recombineering. WT-C (the recombineering construct) should not be confused with the recombineering target WT-P (DHC 64C^wt in attB-Pacman).

^bBecause different numbers of colonies were screened in the various experiments, efficiency is shown as % FL (full length), where FL indicates that the clone passed all screening tests (PCR confirmation with all “check” primer pairs and restriction digest of the full length plasmid). The percentage of wild-type and mutant clones is calculated from full length clones only (e.g., %WT, number of full length clones with wild-type sequence in M segment/number of full length clones).