Joyce
Purify the PCR product with the High Pure PCR Purification kit from Roche
PhybB-RBS-GFP-DT
Digestion mix:
18μl buffer
4.5μl EcoRI
4.5μl SpeI
49μl MQ water
Vector mix:
18μl buffer
4.5μl EcoRI
4.5μl XbaI
49μl MQ water
Ligation:
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
7μl insert
3μl vector
7μl MQ water
Ligate 2 hours at room temperature
Transformation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
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