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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 Run gel 1.3.1.1 Transformation of Biobricks 1.3.2 Extraction PCR 1.4 Results 1.5 Comments

208

Main purpose

Run gel with 9 PCR samples of Nir operon from Pseudomonas aeruginosa

Transformation of Biobricks

PCR reaction for Nir operon

Who was in the lab

Ariadni,Henrike,Julia,Natalia

Procedure

Run gel

• 6 samples from PCR of 09.07.2013
• ladder broadband
• 3 samples from purification of 09.07.2013

Transformation of Biobricks

According to the iGEM protocol ([http://parts.igem.org/Help:Protocols/Transformation transformation protocol]) with slight changes.

• step 1: 100 μl cells
• step 2: 1.5 μl of the resuspended DNA
• step 6: 90 sec (instead of 60 s)at 42 ^oC
• step 9: Incubation without shaking

Transformation list

BioBrick number	Backbone	Resistance	Plate	Well	Copies
98	2:00	1
98	0:20	35
66	0:45	35
72	2:00/3:00/4:00	35
72	5:00	1
4	∞	-

Extraction PCR

9 samples from culture pAO1 10 reactions

• dNTP: 1 ul (10 ul)
• HF buffer: 10 ul (100 ul)
• Phusion polymerase: 0.5 ul (5 ul)
• H₂O: 31.5 ul (315 ul)

Settings for PCR with three different elongation times

Temperature (oC)	Time (min)	Rounds
98	2:00	1
98	0:20	35
66	0:45	35
72	2:00/3:00/4:00	35
72	5:00	1
4	∞	-

Results

Comments

Kanamycin plates were of bad quality

Tetracyclin plates were made by adding 22.5 uL of tetracyclin stock to LB plates

• Stock tetracyclin: 10 mg/mL
• Final concentration in plates: 15 ug/mL