User:JeffreyLau/Notebook/2006-6-15

2006/06/15

Ligation

• Since our gel from yesterday showed failure, we ligated DNA extract from David and Peng.

1. Mixed 6 µL insert (E0241), 2 µL vector (R0010), 2 µL DNA dilution buffer in reaction vial
2. Added 10 µL ligation buffer
3. Added 1 µL ligase, mixed
4. Let incubate 5min at room temperature

Transformation

1. Mixed 20 µL of ligation product with 30 µL competent cells
2. Negative control: 2 µL of dH20 with 30 µL competent cells
3. Positive control: 5 µL of positive control with 30 µL competent cells
4. Incubate on ice for 20min
5. Heat shock @42C for 30s
6. Rest on ice for 2min
7. Incubate overnight @37C