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From 2012.igem.org

Restriction

Ligation

1. Combination of 50 ng of vector with a 2-fold molar excess of insert.
2. Addition of an appropriate volume of T4 Buffer
3. Addition of 0.5-1 µl T4 ligase
4. Adjustment of volume to 20 µl with ddH2O
5. Incubation over night at 16 °C
6. Deactivation of ligase for 10 min at 65 °C

Chemical transformation

1. Thawing of competent cells on ice
2. Addition of 1 µL DNA
3. Incubation on ice for 20 min
4. Heat shock for 1 min at 42 °C
5. Incubation on ice for 5 min
6. Addition of 270 µL LB
7. Incubation in thermoblock for 45 min (incubation time dependent on used vector!) at 37 °C and 300 rpm
8. Smearing of cells on plates and incubation at 37°C (centrifuge it, discard supernatant, resuspend cells in rest-medium, 20 µL on plate)