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:7/30/2014)... 2014 Immunology Summit-2014 ... Principle Investigators, scientists in the field, which includes ... at Case Western Reserve University School of Medicine, ... on “The effects of recombinant human IL-6 ... metalloproteinase-9”. Arya Biragyn, a Senior investigator at National ...
(Date:7/29/2014)... SoundConnect , an award ... proud to announce the strategic additions of implementation, ... Boston, Charleston and Atlanta offices. SoundConnect’s organizational ... for hosted solutions, collaboration and mobility that improve ... , SoundConnect was founded in 2004, with the ...
(Date:7/29/2014)... a number of significant technological challenges in their ... originally created for the James Webb Space Telescope. ... Moseley, a scientist at NASA,s Goddard Space Flight ... actuated microshutter arrays that is, those activated ... functional as the current technology,s magnetically activated arrays. ...
(Date:7/29/2014)...   Sequenom, Inc. (NASDAQ: SQNM ... analysis solutions, and Mayo Medical Laboratories (MML), ... the United States , have announced a ... "We have great appreciation for Mayo Clinic,s ... welcome the opportunity to partner with the organization,s leading ...
Breaking Biology Technology:Unveiling Breakthroughs in Clinical & Cellular Immunology 2Unveiling Breakthroughs in Clinical & Cellular Immunology 3SoundConnect Organization and Culture is Evolving 2Revolutionary microshutter technology hurdles significant challenges 2Revolutionary microshutter technology hurdles significant challenges 3Revolutionary microshutter technology hurdles significant challenges 4Sequenom Enters Into License Agreement With Mayo Medical Laboratories 2Sequenom Enters Into License Agreement With Mayo Medical Laboratories 3Sequenom Enters Into License Agreement With Mayo Medical Laboratories 4
... Tiger and Jacky tip shapes,stand out in Terumo,s newest ... Terumo Interventional Systems announces the launch of Optitorque(TM), ... Tiger and,Jacky tip shapes designed and cleared specifically for ... "The Optitorque catheters, specially designed for radial approach, will,help ...
... biofuel market is estimated at 33 billion with a double digit ... biodiesel. Bioethanol is produced and consumed mainly in Brazil and North ... biodiesel production and this fuel represents about ¾ of the European ... ...
... PLYMOUTH MEETING, Pa., Dec. 1 /PRNewswire-Asia-FirstCall/ -- BMP Sunstone,Corporation ... "Company") today announced,that the company will attend the 20th ... The 20th Annual Piper Jaffray Health ... to Wednesday, December 3, 2008, at the New York,Palace ...
Cached Biology Technology:Terumo Interventional Systems Launches a Complete Line of Coronary Diagnostic Catheters 2Terumo Interventional Systems Launches a Complete Line of Coronary Diagnostic Catheters 3CBDM.T, the Market and Business Intelligence Company Reviews the Biofuel Market 2CBDM.T, the Market and Business Intelligence Company Reviews the Biofuel Market 3CBDM.T, the Market and Business Intelligence Company Reviews the Biofuel Market 4
(Date:7/30/2014)... The Jackson Laboratory has received a pledge of $1 ... and name the Laboratory,s new Neurobehavioral Biometry Center. ... Biometry Center will enable the Laboratory to develop a ... with the ultimate goal of accelerating the pace of ... will serve as a premier resource for the Laboratory ...
(Date:7/30/2014)... Soon, protection from HIV infection could be as simple ... sex. , University of Washington bioengineers have discovered ... that protects women from contracting HIV. Their method spins ... in contact with moisture, releasing higher doses of the ... gels or creams. , "This could offer women a ...
(Date:7/30/2014)... , July 30, 2014 ... mitigation and liveness detection solutions for the biometric ... an industry consortium revolutionizing online authentication with standards ... Mark Cornett , "The decision to join FIDO ... biometric authentication pervasive in use. We believe the ...
Breaking Biology News(10 mins):Rossi Foundation pledges $1M for JAX neurobehavioral research center 2Dissolvable fabric loaded with medicine might offer faster protection against HIV 2Dissolvable fabric loaded with medicine might offer faster protection against HIV 3NexID Biometrics Joins the FIDO Alliance 2
... Mass., May 5 Aware, Inc. (Nasdaq: AWRE ... today reported financial results for its first quarter ended March ... were $4.6 million, a decrease of 22% compared to $5.9 ... its net income and basic and diluted net income per ...
... influenza infection severity have been identified from past outbreaks. ... described in the open access journal BMC Microbiology ... Jonathan Allen and Tom Slezak from Lawrence Livermore National ... acid markers from past pandemic flu strains two weeks ...
... Ind. - Researchers at Purdue University have found a ... in searches for specific genes that shed light on ... Andrew DeWoody, a professor of genetics, and postdoctoral ... step called normalization is no longer necessary with recent ...
Cached Biology News:Aware, Inc. Reports First Quarter 2009 Financial Results 2Aware, Inc. Reports First Quarter 2009 Financial Results 3Aware, Inc. Reports First Quarter 2009 Financial Results 4Aware, Inc. Reports First Quarter 2009 Financial Results 5Aware, Inc. Reports First Quarter 2009 Financial Results 6Random picks better than complicated process in gene identification 2
Annexin V Binding Buffer, 10X Concentrate Buffer , 50 ml Consult technical datasheet for details....
...
IHC Select Anti-p53, prediluted,wild & mutant, a.a. 19-26, clone DO-7 Immunogen: Human p53 Available Date: 37775...
Tube-O-DIALYZER Floats, small....
Biology Products: