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
	control	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 E. coli DH5 alpha competent cells

- 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

Team:Paris/August 29

From 2008.igem.org

Contents

Cloning of EnvZ*

Concentration measurement

Ligation and transformation

Transformation of E. coli DH5 alpha competent cells