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From 2012.igem.org

I. Dilute primers to 10 uM working stock

• Mix:

• 2 uL 200 uM primer stock
• 38 uL sterile, DNAse-, RNAse-free dH₂O

• Note: I think that the p5 primers got mixed up for forward and reverse, but it should not make a difference for the PCR

II. PCR

• Mix Reagents:

• Mix according to the following table, where 1 = pT5, 2 = LacZ, 3 = CAT*, 4 = tet^r, 5 = T1, 0.1 = +template, and 0.2 = -template

• For example, 1.1 = pT5 sample, 1.2 = pT5 control

	1.1	1.2	2.1	2.2	3.1	3.2	4.1	4.2	5.1	5.2
Reagent	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)	(uL)
water	32.5	33.5	33.5	33.5	33.5	33.5	32.5	33.5	32.5	33.5
5x Phusion HiFi buffer	10	10	10	10	10	10	10	10	10	10
10 mM dNTP mix	1	1	1	1	1	1	1	1	1	1
10 uM primer (F)	2.5 (p1f)	2.5 (p1f)	2.5 (p2f)	2.5 (p2f)	2.5 (p3f)	2.5 (p3f)	2.5 (p4f)	2.5 (p4f)	2.5 (p5f)	2.5 (p5f)
10 uM primer (R)	2.5 (p1r)	2.5 (p1r)	2.5 (p2r)	2.5 (p2r)	2.5 (p3r)	2.5 (p3r)	2.5 (p4r)	2.5 (p4r)	2.5 (p5r)	2.5 (p5r)
Template	1 (pBAV1K-T5-gfp)	0	ECNR2 colony*	0	ECFI5 colony*	0	1 (pBR322)	0	1 (pBAV1K-T5-gfp)	0
Phusion HiFi polymerase	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	∞*

*After the cycle ended I moved the tubes to the deli refrigerator for the night

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