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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 (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
J04450	pSB3C5	CAM	2	4D	10-12
J04450	pSB4C5	CAM	2	4F	5
J04450	pSB4K5	Kan	2	6H	5
J04450	pSB1T3	Tetra	2	8B	100-300
J04450	pSB3T5	Tetra	2	8D	10-12
J04450	pSB1AK3	Amp+Kan	2	12B	100-300
J04450	pSB1A3	Amp	2	2H	100-300
J04450	pSB1A2	Amp	5	1B	100-300
J04450	pSB6A1	Amp	5	1K	10-15
J04450	pSB1K3	Kan	5	5A	100-300
J04450	pSB3K3	Kan	5	5E	10-12
K325909	pSB1C3	CAM	1	4L	100-300
K325209	pSB1C3	CAM	1	12H	100-300
K325219	pSB1C3	CAM	1	2D	100-300
J04421	pSB1C3	CAM	3	15O	100-300
E0430	pSB1C3	CAM	3	20K	10-300

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