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	[[File:2013-07-11_Nir_PCr_results.jpg|600px]]		[[File:2013-07-11_Nir_PCr_results.jpg|600px]]
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		+	==Conclusion==
		+	We have now tried PCR of Nir more than one time and same result appear. This must then be the true chromosomal fragment but with some additional sequence in the middle. We will try to do another PCR with USER primers tomorrow to see if we then get same fragments or shorter ones.

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Revision as of 16:41, 12 July 2013

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 1.3.3 Gel analyses of Nir operon, PCR product from 10-07-2013 1.3.4 PCR purification 1.4 Result 1.5 Conclusion

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.

Who was in the lab

Ariadni, Henrike, Julia, Jakob, Gosia

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 1(a, b, c)
• Nir 2(a, b, c)

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 AraBAD promoter using plasmid pZA21 without its native promoter and RFP gene. We performed 3 different PCR reactions using user primers. Each PCR reaction was performed in triplicate.

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 -> AraBAD 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	∞	-

Gel analyses of Nir operon, PCR product from 10-07-2013

We run 0,8% agarose gel with 9 samples.

PCR purification

PCR fragments for the Nir operon from todays PCR.

Result

0.8 % Agorase Gel (Nir operon)

Wells

• 1: Nir extension time 2:00
• 2: Nir extension time 2:00
• 3: Nir extension time 2:00
• 4: Nir extension time 3:00
• 5: 1kb ladder
• 6: Nir extension time 3:00
• 7: Nir extension time 3:00
• 8: Nir extension time 4:00
• 9: Nir extension time 4:00
• 10: Nir extension time 4:00

Conclusion

We have now tried PCR of Nir more than one time and same result appear. This must then be the true chromosomal fragment but with some additional sequence in the middle. We will try to do another PCR with USER primers tomorrow to see if we then get same fragments or shorter ones.