Equipment

 • Shaking incubator at 37 °C 

 • Stationary incubator at 37 °C

 • Water bath at 42 °C

 • Ice bucket filled with ice

 • Microcentrifuge tubes 

 • Sterile spreading device 

 

Reagents 

 • LB agar plate (with appropriate antibiotic) 

 • LB or SOC media

 • Competent cells 

 • DNA you'd like to transform

 

 

 

 

Procedure 

 

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). 

 

 

2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator. 

 

 

3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times. 

 

Note) Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid. 

 

 

4. Incubate the competent cell/DNA mixture on ice for 20-30 mins. 

 

 

5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using). 

 

 

6. Put the tubes back on ice for 2 min. 

 

 

7. Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min. Note) This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances. 

 

 

8. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic. 

 

 Note) We recommend that you plate 50 μL on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.

 

If the culture volume is too big, gently collect the cells by centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before the cells begin dividing, the bacteria diffuse through the liquid and won't grow in colonies.

 

 

9. Incubate plates at 37°C overnight.