Team:Paris/August 29

From 2008.igem.org

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(→Cloning of EnvZ*)
(→Transformation of E. coli DH5 alpha competent cells)
 
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==Ligation and transformation==
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==Ligation==
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*10 min at room temperature
*10 min at room temperature
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*7 hours at about 16°C in the room tagged "danger azote liquide"
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*followed by 7 hours at about 16°C.
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==Transformation==
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==Transformation of E. coli DH5 alpha competent cells==
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<br>- defrosing of the cells on ice
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* Defroze of the cells on ice
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<br>- adding of 5 µL of ligation products in 100 µL of competent cells in 14 mL falcon polypropylene tubes
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* Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
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<br>- 30 min on ice
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* 30 min on ice
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<br>- thermal schock of 45 s at 42°C
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* Heat schock 45sec at 42°C
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<br>- 2 min on ice
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* 2 min on ice
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<br>- adding of 900 µL of SOC
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* Add 900 µL of SOC
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<br>- contrifugation 5 min 6000 rpm
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* Incubate 1h at 37°C
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<br>- removal of 800 µL
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* Centrifugate 5 min at 6000 rpm
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<br>- plating the 200 µL left on LB + amplicilline
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* Remove 800 µL
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<br>- overnight incubation at 37°C
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* Plate the 200 µL left on LB + amplicilline
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* Incubate O/N at 37°C

Latest revision as of 18:55, 16 September 2008

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Contents

Cloning of EnvZ*

Concentration measurement

DNA concentration A260 / A2801
D159 (EnvZ*) 9 µg/mL 1,59
D116 (pSB1A2) 13 µg/mL 1,29

Ligation

control insert / vector ratio
3 / 1 4 / 1
D159 (EnvZ*) 0 8,7 µL 11,6 µL
D116 (pSB1A2) 2 µL 2 µL 2 µL
T4 DNA ligase 10X buffer 2 µL 2 µL 2 µL
T4 DNA ligase 1 µL 1 µL 1 µL
H2O (qsp 20 µL) 15 µL 6,3 µL 3,4 µL
  • 10 min at room temperature
  • followed by 7 hours at about 16°C.

Transformation of E. coli DH5 alpha competent cells

  • Defroze of the cells on ice
  • Add 5 µL of DNA in 100 µL of competent cells (ligation products or pUC19 for positive control)
  • 30 min on ice
  • Heat schock 45sec at 42°C
  • 2 min on ice
  • Add 900 µL of SOC
  • Incubate 1h at 37°C
  • Centrifugate 5 min at 6000 rpm
  • Remove 800 µL
  • Plate the 200 µL left on LB + amplicilline
  • Incubate O/N at 37°C
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