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Bacterial Transformation (KPY)
Following 10 μL Ligation With NEB T4 DNA Ligase
- Chemically Competent E. Coli Cells
- 10 μL Ligation Mixture
- Retrieve chemically competent cells from -80°C freezer.
- Thaw cells for 5 minutes on ice.
- Add 2.5 μL of 10 μL ligation mixture into the microcentrifuge tubes containing the cells, stir gently, and then agitate the tubes.
- Ice the cells for 30 minutes.
- Heat shock the cells for 5 minutes at 37°C in the water bath.
- Incubate the cells on ice for 2 minutes.
- Add 500 μL of LB (no antibiotic) to the cells.
- Place the cells back into water bath for another 30 minutes of incubation.
- Spread the mixture of LB and cells onto a plate with the appropriate antibiotic.
- Grow overnight at 37°C.
- Incubation time on ice can be reduced to 15 minutes in case of rush.
- Water bath incubation can also be reduced to 15 minutes in case of rush.
- Mid-October ligation/transformation failures were resolved with a change of loading dye following enzymatic digestion of DNA.
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