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From 2012.igem.org

I. Cross-over PCR

• Template Mix Specifications:

Template	Size (bp)	Amount Wanted (pmol)	Amount Wanted (ng)	Concentration (ng/uL)	Volume (1x)	Volume (5x)
LacZ	3151	0.00745	15.24	11.468	1.33	6.7
CAT*	729	0.00745	3.52	35.256	0.1	0.5
tetR	1265	0.00745	6.12	37.787	0.16	0.8
total						8 uL

• *Calculations made with [http://molbiol.edu.ru/eng/scripts/h01_07.html this website]

• Mix Reagents:

• Mix according to the following table

	1 Reaction	Master Mix (x6)
Reagent	(uL)	(uL)
water	31.9	191.4
5x Phusion HiFi buffer	10	60
10 mM dNTP mix	1	6
10 uM primer (F)	2.5 (pBf)	15 (pBf)
10 uM primer (R)	2.5 (pBr)	15 (pBr)
Template Mix	1.6	-
Phusion HiFi polymerase	0.5	3

• Before adding template, move 48.4 uL of Master Mix to a clean PCR tube and add 1.6 uL water for negative control
• Add templates to Master Mix according to Template Specifications above, mix by pipetting
• Aliquot 50 uL of mix to 5 PCR tubes

• Note: Last tube had less than others

• Run PCR O/N

Step	Temp (oC)	Time
1	94	4 m
2	94	30 s
3	54	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	∞*