Team:DTU-Denmark/Notebook/10 July 2013

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10 July 2013

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Contents

Lab 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

  • 3 samples from PCR on the 09.07.2013 using PAO1 as template source
  • 3 samples from PCR on the 09.07.2013 using PCR purification of 08.07.2013 as template source
  • ladder broad range biggest fragment = 20 kb
  • 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

0.8 % Agorase Gel (Nir operon)

Wells

  • 1: Nir, yesterday's PCR, source: culture PAO1
  • 2: Nir, yesterday's PCR, source: culture PAO1
  • 3: Nir, yesterday's PCR, source: culture PAO1
  • 4: Nir, yesterday's PCR, source: purification from 09.07.2013
  • 5: Nir, yesterday's PCR, source: purification from 09.07.2013
  • 6: Nir, yesterday's PCR, source: purification from 09.07.2013
  • 7: Broadband Ladder
  • 8: Nir purification of 09.07.2013
  • 9: Nir purification of 09.07.2013
  • 10: Nir purification of 09.07.2013

2013 07 10 gel nir.jpg

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

Conclusions

Nir forms a nice, but weak, band in most samples but the fragments are still far longer than expected. It looks like they are somewhere around 16 kb where we rather expected 9102 bp. We will try to cut down the extension time and see if we can get something out of it.


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