Team:DTU-Denmark/Notebook/10 July 2013

From 2013.igem.org

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(→Transformation of Biobricks)
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Line 29: Line 29:
! BioBrick number!! Backbone!! Resistance!! Plate!! Well!! Copies
! BioBrick number!! Backbone!! Resistance!! Plate!! Well!! Copies
|-
|-
-
| 98 || 2:00 || 1
+
| J04450 || pSB3C5 || CAM || 2 ||4D || 10-12
|-
|-
-
| 98 || 0:20 || 35
+
| J04450 || pSB4C5 || CAM || 2 ||4F || 5
|-
|-
-
| 66 || 0:45 || 35
+
| J04450 || pSB4K5 || Kan || 2 ||6H || 5
|-
|-
-
| 72 || 2:00/3:00/4:00 || 35
+
| J04450 || pSB1T3 || Tetra || 2 ||8B || 100-300
|-
|-
-
| 72 || 5:00 || 1
+
| J04450 || pSB3T5 || Tetra || 2 ||8D || 10-12
|-
|-
-
| 4|| ∞ || -
+
| 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
 +
 
|}
|}

Revision as of 16:57, 10 July 2013

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
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