Kai Yuet/Protocols:Transformation

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Contents

Bacterial Transformation (KPY)

Following 10 μL Ligation With NEB T4 DNA Ligase

Materials

Procedures

  1. Retrieve chemically competent cells from -80°C freezer.
  2. Thaw cells for 5 minutes on ice.
  3. Add 2.5 μL of 10 μL ligation mixture into the microcentrifuge tubes containing the cells, stir gently, and then agitate the tubes.
  4. Ice the cells for 30 minutes.
  5. Heat shock the cells for 5 minutes at 37°C in the water bath.
  6. Incubate the cells on ice for 2 minutes.
  7. Add 500 μL of LB (no antibiotic) to the cells.
  8. Place the cells back into water bath for another 30 minutes of incubation.
  9. Spread the mixture of LB and cells onto a plate with the appropriate antibiotic.
  10. Grow overnight at 37°C.

Notes

  • 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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