( ...)Escherichia coli DH10B

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.513 04/2002 Microorganism Escherichia coli DH10B Cell type Bacteria, gram negative Molecules injected Plasmid DNA (pUC 19) Growth medium LB medium Washing solution Sterile, ice-cold water; (10% glycerol) Electroporation solution Sterile, ice-cold water; (10% glycerol) Outgrowth medium SOC medium (without antibiotics) Cuvette 1 mm gap width Reference Eppendorf AG Application Hotline D-22331 Hamburg
Phone +49 180 3 666 789 Fax +49 40 53990 125 e-mail: application-hotline@eppendorf.de
Adapted from: High Efficiency Transformation by Electroporation Short Protocols in Molecular Biology
Second Edition Green Publishing Association and John Wiley & Sons, New York 1-22 1-23 Making electrocompetent cells:

1. Inoculate 500 ml LB medium with 2.5 ml of a fresh overnight culture of E. coli DH10B. Grow at 37 C with shaking to an O.D.₆₀₀ of 0.5 to 0.6. 2. Chill cells on ice for 15 minutes and transfer to a prechilled centrifuge bottle. Harvest by centrifugation (20 minutes, 5,000 x g, 2-4 C). Resuspend pellet in 5 ml ice-cold water. Keep the cells cold during the entire procedure. 3. Wash twice with the original culture volume of ice-cold water. Centrifuge as above. Resuspend pellet by swirling in remaining liquid. 4a. If using the cells immediately, place suspension in a prechilled tube and centrifuge (10 minutes, 5,000 x g, 2-4 C). Resuspend the cells in ice-cold water to a final concentration of approximately 2 x 10¹¹ cells/ml. Aliquote 40-300 l cells into prechilled centrifuge tubes. 4b. If freezing the cells for later use, add 40 ml of ice-cold 10% glycerol, mix and centrifuge for 10 minutes, at 5,000 x g and 2-4 C. Resuspend the cells in ice-cold 10% glycerol to a final concentration of approximately, 2 x 10¹¹ cells/ml. Aliquote 40-300 l cells into prechilled centrifuge tubes. quick freeze on dry ice and store at 80 C.

Electroporation of cells:

1. Add 1 l DNA (10 pg in water) to tubes containing 40 l electrocompetent cells. Homogenize by gently mixing with pipette several times.
2. Transfer mixture to a prechilled cuvette. Wipe moisture from the cuvette and insert the cuvette into the device.
3. Electroporation:
   
   Mode Prokaryotes O Voltage (V) 1,660 V Time constant (T) 5 ms
   
4. Immediately add 1 ml SOC medium and transfer to a sterile culture tube with a pasteur pipette. Incubate 30-
   60 minutes with moderate shaking at 37 C.
5. Plate on LB plates containing the appropriate selection chemical.

Expected Results: Transformation efficiency up to 4 x 10⁹ transformants/g of DNA.


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