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From 2012.igem.org

I. PCR

• Mix Reagents:

• Mix according to the following table, where 2 = LacZ, 3 = CAT*, 4 = tet^r, 0.1-0.3 = +template, and 0.4 = -template

• For example, 2.1 = lacZ sample, 2.4 = lacZ control

	2.1	2.2	2.3	2.4	3.1	3.2	3.3	3.4	4.1	4.2	4.3	4.4
Reagent	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)
water	33.5	33.5	33.5	33.5	33.5	33.5	33.5	33.5	32.5	32.5	32.5	33.5
5x Phusion HiFi buffer	10	10	10	10	10	10	10	10	10	10	10	10
10 mM dNTP mix	1	1	1	1	1	1	1	1	1	1	1	1
10 uM primer (F)	2.5 (p2f_2)	2.5 (p2f_2	2.5 (p2f_2)	2.5 (p2f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p3f_2)	2.5 (p4f)	2.5 (p4f)	2.5 (p4f)	2.5 (p4f)
10 uM primer (R)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p2r_2)	2.5 (p3r)	2.5 (p3r)	2.5 (p3r)	2.5 (p3r)	2.5 (p4r_2)	2.5 (p4r_2)	2.5 (p4r_2)	2.5 (p4r_2)
Template	ECNR2 colony*	ECNR2 colony*	ECNR2 colony*	0	ECFI5 colony*	ECFI5 colony*	ECFI5 colony*	0	1 (pBR322)	1 (pBR322)	1 (pBR322)	0
Phusion HiFi polymerase	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5

*For these templates, I touched a colony with a sterile pipette tip and spread it on the bottom of the PCR tube before adding reagents

• Run PCR

Step	Temp (oC)	Time
1	94	4 m
2	94	30 s
3	53	30 s
4	72	1.5 m
5	GOTO 2	7x
6	94	30 s
7	66	30 s
8	72	1.5 m
9	GOTO 6	30x
10	72	5 m
11	4	∞*

• Store tubes on ice after PCR is finished