Team:Paris/August 29

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(→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 "danger azote liquide"
+
*7 hours at about 16°C in the room tagged "danger azote liquide"
*transformation of 100 µL of competent E. coli DH5 alpha with 5 µL of ligation product (thermal schock of 45 s at 42°C)
*transformation of 100 µL of competent E. coli DH5 alpha with 5 µL of ligation product (thermal schock of 45 s at 42°C)
*plating on LB + amplicilline
*plating on LB + amplicilline
*overnight incubation at 37°C
*overnight incubation at 37°C

Revision as of 17:54, 29 August 2008

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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 of 100 µL of competent E. coli DH5 alpha with 5 µL of ligation product (thermal schock of 45 s at 42°C)
  • plating on LB + amplicilline
  • overnight incubation at 37°C
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