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From 2013.igem.org

Contents 1 208 1.1 Main purpose 1.2 Who was in the lab 1.3 Procedure 1.3.1 Combine Nir operon with pZA21 1.3.2 PCR for promoter test

208

Main purpose

• PCR with USER primers to combine Nir operon with pZA21

• PCR with USER primer to test the activity of the promoter AraB. PCR for promoter test

Who was in the lab

Ariadni,Henrike,Julia,Jakob

Procedure

Combine Nir operon with pZA21

Following Mathildes Protocol for DpnI digestion of our linear pZA21 w. Uracil incerts

Mastermix for 7 PCR tubes, Using the mastermix protocol we got from Mathilde.

triplets of each of the two Nir Operon extracts that we have made yesterday, including one negative control:

• Nir 2a(I,II,III)
• Nir 2b(I,II,III)

Using the following PCR program:

	time	Temp C
Denaturing	2 min	98
start of loop	10 sec	98
annealing	30 sec	60
elongation	9 min	72
goto start of loop 35 times
hold		10

PCR for promoter test

We want to test the inducible AraB promoter using plasmid pZA21 without its native promoter and RFP gene. We performed 3 different PCR reactions using user primers. Samples are named:

• 1,2,3 -> pZA21 with no native promoter - Primers 13a and 13b, template pZA21 miniprep
• 4,5,6 -> RFP - Primers 14a and 14b, template RFP in pZE21 miniprep
• 7,8,9 -> AraB promoter - Primers 12a and 12b, template biobrick K808000

Settings for PCR of 1,2,3

Temperature (oC)	Time (min)	Rounds
98	2:00	1
98	0:20	35
58	0:45	35
72	2:00	35
72	5:00	1
10	∞	-

Settings for PCR of 4-9

Temperature (oC)	Time (min)	Rounds
98	2:00	1
98	0:20	35
61	0:45	35
72	1:00	35
72	5:00	1
10	∞	-