Navigation Links
Escherichia coli DH5

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.512 04/2002 Microorganism Escherichia coli DH5alpha 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 DH5alpha. 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) 1,700 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 3 x 109 transformants/g of DNA.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. Escherichia coli C600
2. Escherichia coli DH10B
3. Escherichia coli DH5
4. Escherichia coli K12
5. Escherichia coli C600
6. Escherichia coli K12
7. Escherichia coli DH10B
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: Escherichia coli

(Date:1/22/2015)... 22, 2015 Shimadzu Scientific Instruments ... multipurpose UV-visible spectrophotometer. Offering wavelength scanning from 190 ... ideal for applications in a variety of industries, ... The user-friendly UV-1280 enables intuitive operation, while the ...
(Date:1/22/2015)... Cambridge Semantics, the leading provider of smart data solutions ... a record-breaking year across the board for the company. ... Platform and our Smart Data solutions, our customers were able ... led to record growth for the company in 2014,” said ...
(Date:1/22/2015)... Diego, California (PRWEB) January 22, 2015 ... Promotional Offer to their comprehensive portfolio of Eppendorf ... allows customers to purchase a Centrifuge 5424/5424 R and ... Bundle of either Eppendorf Research plus or Eppendorf Reference ...
(Date:1/22/2015)... 2015 Selexis SA, a serial innovation ... Banks (RCBs) used for drug discovery to commercial manufacturing, ... include Next-Generation Sequencing (NGS) data packages. The ... manufacturing by ensuring the integrity of the gene, validation ...
Breaking Biology Technology:Shimadzu’s New Monitored Single-Beam UV-Vis Spectrophotometer Offers Comprehensive Measurement Options in a Compact Body 2Cambridge Semantics Announces Record Results for 2014 2Eppendorf Announces Promotional Bundle Which Includes: Eppendorf Centrifuge 5424, 3-Pack of Pipettes, and Tips - Available Now at Pipette.com 2Selexis Generated Research Cell Banks Now Fully Sequenced Using Next-Generation Sequencing 2
... May 7 Dendreon Corporation (Nasdaq: DNDN ) today announced ... Bank of America and Merrill Lynch 2009 Health Care Conference, ... May 12, 2009, 9:20 a.m. ET, ... Tuesday, May 19, 2009, 4:35 p.m. ...
... Thursday, May 7, 2009 at 4:30 p.m. Eastern ... OncoGenex Pharmaceuticals, Inc. ("OncoGenex" or the "Company") (NASDAQ: ... results for the three months ended March 31, ... first quarter of 2009. , The following consolidated ...
... 6 Spectros Corporation (Portola Valley, CA) filed ... Scientific (NYSE: TMO ), alleging infringement ... 3300 product. Spectros invented and commercialized solid-state, broadband, ... and incorporated these into its flagship T-Stat product, ...
Cached Biology Technology:OncoGenex Reports First Quarter Financial Results 2OncoGenex Reports First Quarter Financial Results 3OncoGenex Reports First Quarter Financial Results 4OncoGenex Reports First Quarter Financial Results 5OncoGenex Reports First Quarter Financial Results 6Spectros Corporation Protects Its Cool White LED Spectroscopy Technology Against Alleged Infringement by Thermo Fisher Scientific 2
(Date:1/22/2015)... ("Company") (BM&FBOVESPA: VLID3 ON), is pleased to inform its shareholders ... Washington,s Department of Licensing, in the United States, awarded ... for driver,s licenses and identification cards to its subsidiary, Valid ... start in January 2015, with enrollment and card production starting ...
(Date:1/22/2015)... , Jan. 13, 2015  Today, FreeWavz ( ... launched its crowdfunding campaign on Fundable, https://www.fundable.com/freewavz ... build of production capacity to meet customer demand. ... Photo - http://photos.prnewswire.com/prnh/20150113/168790 ...
(Date:1/22/2015)...  BellBrook Labs, a leader in high throughput screening ... a TR-FRET (time resolved Forster resonance energy transfer) ... high throughput screening assay for glycosyltransferases and other ... sensitive detection of hundreds of human and microbial ...
Breaking Biology News(10 mins):Valid USA Signs Contract For Washington Driver's License Issuance 2FreeWavz Launches on Fundable to Drive Speed-to-Market in 2015 2BellBrook Labs Launches a High Throughput Screening Assay for Glycosyltransferases with TR-FRET Readout 2
... Institute researcher Dr Matthew Call has been awarded ... Innovation (VESKI) Fellowship by the Victorian Government to ... and signalling. Dr Call,s research could signal ... treating immune diseases, using creative ,membrane mimic, technology ...
... born to mothers who are fed a diet supplemented with ... scientists at the Jean Mayer USDA Human Nutrition Research Center ... research in humans and mice suggests that B vitamins, particularly ... Using a mouse model of naturally occurring colorectal cancer, the ...
... Ariz. June 9, 2011 TGen Drug Development ... the rapid development of anti-cancer drugs and deliver them ... "The highest quality medical imaging provided by Imaging ... Dr. Stephen Gately, President of TD2, which assists drug ...
Cached Biology News:Structural biologist wins $150K for 'membrane mimic' technology 2B vitamins in mother's diet reduce colorectal cancer risk in offspring 2TGen Drug Development partners with Imaging Endpoints for comprehensive clinical trial services 2
... produced by a patented microwave process which ... consistent, even cell attachment, increased cell growth ... growth uunder difficult conditions, including reduced serum ... a more ecominic alternative to expensive and ...
Automated Cell Culture Flask...
... Bio-Plex suspension array system, 100-240 V, uses ... up to 100 proteins and peptides in ... composed of up to 100 color-coded bead ... with a unique reactant (enzyme substrates, receptors, ...
Cell Culture Flask, 75 cm, treated polystyrene...
Biology Products: