advertisement
Advertisement (image not found)

Salmonella Typhimurium

ShareThis

Salmonella typhimurium
Multiporator / Electroporator 2510
Transformation Protocol Protocol No. 4308 915.532 – 01/2002
Microorganism Salmonella typhimurium LT2
Cell type Bacteria, gram negative
Molecules injected Plasmid DNA (pBR322 in TE buffer)
Growth medium LB medium
Washing solution 10 mM HEPES, pH 7.0; 10% glycerol
Electroporation solution 10% glycerol
Outgrowth medium SOC medium (without antibiotics)
Cuvette 2 mm gap width
Reference Binotto J., et al • 1991 • Canadian Journal of Microbiology 37 • 474-477
Making electrocompetent cells:

1. Incoculate a flask of LB 1:100 with a fresh overnight culture, grow at 37 °C with shaking to an O.D.640 of 0.75. Chill the cells in an ice-water bath for 15 min.
2. Harvest by centrifugation (4,000 x g, 10 min., 4 °C).
3. Wash one time in the original culture volume with HEPES and one time with 10% glycerol using 1/100 of the original volume.
4. Resuspend in 10% glycerol.

Electroporation of cells:

  1. Add 2 µl plasmid DNA (20 pg to 20 ng) to 40 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes “O“
    Voltage (V) 2,400 V
    Time constant (T) 5 ms

  4. Immediately add 1 ml SOC medium and transfer to a sterile culture tube. Incubate for 1 hour at 37 °C with shaking.
  5. Dilute the cells in SOC medium and plate on selective LB plates.
Expected Results:
Transformation efficiency up to 4 x 108 transformants/µg of DNA.

Contact Information

In the United States:
Eppendorf North America, Inc.
102 Motor Parkway,
Hauppauge, NY 11788-5178
Tel: 800-645-3050
Fax: 516-334-7506
Web Site: http://www.eppendorfna.com/

Outside the United States:
Eppendorf AG
Barkhausenweg 1
22339 Hamburg
Germany

Customer Service: ++ 49 40 53 801-0

Fax Number: ++ 49 40 53 801-556

Article Tools
  • Email a Colleague
  • Print
  • ShareThis
advertisement
Advertisement (image not found)

Email Newsletter Sign-Up

Stay updated on the latest technologies and news with Biocompare's newsletters
(See samples here)












 Select All

Privacy Policy | Manage Subscriptions

Loading

Loading