METHODBOOK

Transformation of plasmid DNA to competent E. Coli cells

Chenzhong Kuang

Ver.1.0 - 7/7/2002

Material and Reagents

1. SOC

   2% Tryptone
   0.5% Yeast Extract
   10mM NaCl
   10mM MgSO₄
   10mM MgCl₂

2. 1.5 mL microfuge tubes

3. 42°C waterbath

4. Ice

5. 37°C shaker

Protocol

1. Thaw competent cells on ice. 20-200μL per tube

2. Add max. 20μL of a ligation reaction

3. Mix very gently!

4. Incubate the tubes on ice for 30 min

5. Heat shock the cells for 45 sec to 2 min at 42°C

6. Place the tubes immediately on ice for at least 2 min

7. Add 800μL of SOC medium to each tube

8. Incubate for 1 hour at 37°C and shake vigorously

9. Spin down briefly and remove most supernatants

10. Resuspend cell pellet with the rest SOC medium in the tube by pipetting

11. Plate out the suspension on a LB agar plate containing the appropriate antibiotic.

12. Incubate the plates overnight at 37°C

Remarks

• I have use this protocol to transform DH5 alpha competent cells successfully

• When transforming purified plasmid into competent cells, I just add 1μL plasmid DNA solution. Then plate out only 10-20μL bacterial suspension to the plate instead of all

• Efficiency depends on ligation reaction and competent activity.

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