Team:Paris/August 29

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(Difference between revisions)
(→Cloning of EnvZ*)
(→Cloning of EnvZ*)
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*10 min at room temperature
*10 min at room temperature
*7 hours at about 16°C in the room tagged "danger azote liquide"
*7 hours at about 16°C in the room tagged "danger azote liquide"
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*transformation of 100 µL of E. coli DH5 alpha competent cells with 5 µL of ligation product
+
 
 +
==transformation==
 +
 
<br>- defrosing of the cells on ice
<br>- defrosing of the cells on ice
<br>- adding of 5 µL of ligation products in 100 µL of competent cells in 14 mL falcon polypropylene tubes
<br>- adding of 5 µL of ligation products in 100 µL of competent cells in 14 mL falcon polypropylene tubes

Revision as of 18:02, 29 August 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 and transformation

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
  • 7 hours at about 16°C in the room tagged "danger azote liquide"

transformation


- defrosing of the cells on ice
- adding of 5 µL of ligation products in 100 µL of competent cells in 14 mL falcon polypropylene tubes
- 30 min on ice
- thermal schock of 45 s at 42°C
- 2 min on ice
- adding of 900 µL of SOC
- contrifugation 5 min 6000 rpm
- removal of 800 µL
- plating the 200 µL left on LB + amplicilline
- overnight incubation at 37°C

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