TABLE 1. Colony mutation frequencies for experiments with 3D^pol.

aExp. #	bCondition	White/Faint Blue Colonies	Total Colonies	cColony Mutation Frequency (CMF)	CMF- BKG.
1	Background	49	3609	0.014	0
	0.8 mM MgCl2, 50 μM rNTPs	71	2712	0.026	0.012
	4 mM MgCl2, 500 μM rNTPs	87	2529	0.034	0.020
	0.8 mM MgCl2, 50 μM G, U, CTP, 5	51	1900	0.027	0.013
	μM ATP
	0.8 mM MgCl2, 50 μM G, U, CTP, 0.5	111	2526	0.044	0.030
	μM ATP
2	Background	61	3705	0.016	0
	0.8 mM MgCl2, 50 μM rNTPs	63	2487	0.025	0.009
	4 mM MgCl2, 500 μM rNTPs	75	3023	0.025	0.009
	4 mM MnCl2, 500 μM rNTPs	162	1734	0.093	0.077
3	Background	26	2633	0.010	0
	4 mM MgCl2, 500 μM rNTPs	45	3067	0.015	0.005
	4 mM MgCl2, 500 μM rNTPs +3AB	44	2648	0.017	0.007
4	Background	10	1261	0.008	0
	4 mM MgCl2, 500 μM rNTPs	16	1070	0.015	0.007
	4 mM MgCl2, 500 μM rNTPs +3AB	31	1874	0.014	0.006
5	Background	23	2587	0.009	0
	0.8 mM MgCl2, 50 μM rNTPs	50	2784	0.018	0.009
	4 mM MgCl2, 500 μM rNTPs	50	3467	0.014	0.005
6	Background	11	1427	0.008	0
	0.8 mM MgCl2, 50 μM rNTPs	28	1971	0.014	0.006
	4 mM MgCl2, 500 μM rNTPs	20	1365	0.015	0.007
Average values +/− standard deviation for experiments with multiple repeats
		Ave.		Ave. -BKG.
Background		0.011 +/− 0.003		0
0.8 mM MgCl2, 50 μM rNTPs		0.021 +/− 0.006		0.009 +/− 0.002
4 mM MgCl2, 500 μM rNTPs		0.020 +/− 0.008		0.009 +/− 0.006
4 mM MgCl2, 500 μM rNTPs +3AB		0.016 +/− 0.002		0.007 +/− 0.001

^aIndependent experiments performed at different times.

^bIn background assays the T3 RNA polymerase template RNA was directly reverse transcribed by RT and this cDNA product was used in PCR reactions. Assays to test 3D^pol fidelity had two additional 3D^pol synthesis steps prior to PCR. These included extension of a primer on the original T3 template, recovery of the synthesized product, and a second round of 3D^pol synthesis using this product as the template.

^cNumbers shown are the “colony mutation frequency” (CMF) defined as white + faint blue colonies divided by total colonies. Refer to the Results and Methods sections for details.