Team:DTU-Denmark/Notebook/10 July 2013

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Revision as of 16:57, 10 July 2013 by Ariadni (Talk | contribs)

Contents

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
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