Navigation Links
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:
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.



Page: All 1 2 3

Related biology technology :

1. Escherichia coli C600
2. Escherichia coli DH5
3. Escherichia coli K12
4. Escherichia coli C600
5. Escherichia coli K12
6. Escherichia coli DH10B
7. Escherichia coli DH5
Post Your Comments:

(Date:10/13/2015)... ... 2015 , ... The Pistoia Alliance has appointed Dr Steve ... drive to increase the organization’s impact on the global innovation landscape in life ... its work in supporting pre-competitive collaboration in the life sciences sector. Steve brings ...
(Date:10/13/2015)... Applied BioMath ( ), a computational ... the drug invention process, announced that it will sponsor ... 20-22, 2015 in Boston , MA.  ... CEO of Applied BioMath, will present "QSP approaches enabling quantitative ... trials" on Wednesday October 21st 2015 at 12:30PM.  ...
(Date:10/13/2015)... ARRY ), Avanex Life Sciences ... ), and Progenix Pharmaceuticals, Inc. (NASDAQ: PGNX ) ... (OTCQX: AVXLD), Juno Therapeutics, Inc. (NASDAQ: JUNO ), ... --> Cooperative research and development efforts with the ... to advancements in the treatment of severe wounds.  Biotech ...
(Date:10/13/2015)... and SAN DIEGO , ... Mast Therapeutics, Inc. have agreed to collaborate in the ... poloxamer 188 NF, marketed by BASF under the Kolliphor ... a variety of pharmaceutical and biological applications, such as ... is the starting material for Mast,s lead product candidate. ...
Breaking Biology Technology:
(Date:9/30/2015)... Sept. 30, 2015  With nearly 300,000 Americans living ... new SCIs estimated to reach 12,500 annually, the role ... Resource Services for Independent Living (SCRS-IL) is increasingly ... California opening doors to independence for ... and services, notably assistive technology services and education. ...
(Date:9/29/2015)... Calif. , Sept. 29, 2015 ... improves employee productivity while also saving energy , ... such as Low Power Active Mode and embedded Fujitsu ... transformation Fujitsu today shows that good ... new and refreshed models to its enterprise desktop and ...
(Date:9/28/2015)... 28, 2015  The monitoring of vital signs, ... temperature, is an essential component of patient assessment. ... deterioration in a patient,s condition. However, in general ... typically taken during routine observation rounds only once ... deteriorates between these observation rounds, the warning signs ...
Breaking Biology News(10 mins):