Navigation Links
Azotobacter vinelandii

Multiporator / Electroporator 2510 Transformation Protocol

Protocol No. 4308 915.503 04/2002

Microorganism Azotobacter vinelandii Cell type Bacteria, gram negative Molecules injected Plasmid DNA Growth medium Nitrogen-free medium Washing solution Ice-cold 10% sterile glycerol Electroporation solution Ice-cold 10% sterile glycerol Outgrowth medium Azotobacter growth (AG) medium Cuvette 1 mm gap width Reference Kornyi, P. et al 1998 Research in Microbiology 149 361-372

Making electrocompetent cells:

  1. Cultivate cells with vigorous shaking at 30 C to an O.D.620 of 0.4-0.5.

  2. Harvest by centrifugation at 5,000 rpm for 10 min at 0 C.

  3. Wash with the original culture volume of ice-cold 10% glycerol.

  4. Repeat this step three times using a half and a quarter of the original volume, and finally 2-4 ml of the glycerol solution (7.0 x 107 cells/ml final cell concentration).

  5. Freeze 40 l aliquots in liquid nitrogen and store at 70 C.

Electroporation of cells:

  1. Add 1 l (300 ng/l) plasmid DNA to 40 l of electrocompetent cells. Homogenize by gently mixing with pipette

  2. several times. Transfer mixture into a prechilled cuvette.
  3. Wipe moisture from the cuvette and insert the cuvette into the device.

  4. Electroporation:

  5. Mode Prokaryotes O Voltage (V) 1,800 V Time constant (T)
    5 ms No. of pulses (n) 1
  6. Immediately add 1 ml AG medium and incubate with shaking at 200 rpm for 1 h at 30 C.

  7. Plate onto selective AG media plates.

Expected results:

Transformation efficiency up to 6.8 x 105 transformants/g of DNA.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Azotobacter vinelandii
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/29/2016)... ... April 29, 2016 , ... During a ... technology into a viable company, CereScan’s CEO, John Kelley, joined other Denver business ... leader and mentor in the Denver area business community, shared his top fundamental ...
(Date:4/29/2016)... , ... April 29, 2016 , ... Intelligent Implant Systems ... the FDA via 510(k) for sale in the United States. These components expand ... thoraco-lumbar fusions. With one-level sales beginning in October of 2015, the company has ...
(Date:4/28/2016)... , April 28, 2016 ... Stirling, and Brayton Cryocoolers), Service (Technical Support, Product Repairs ... Geography - Global Forecast to 2022", published by MarketsandMarkets, ... 2.94 Billion by 2022, at a CAGR of 7.29% ... market data Tables and 94 Figures spread through 159 ...
(Date:4/28/2016)... ... April 28, 2016 , ... ... for Connecticut's innovative, growing companies, today announced the launch of VentureClash , ... (fintech) companies. , “VentureClash looks to attract the best early-stage companies ...
Breaking Biology Technology:
(Date:3/31/2016)... , March 31, 2016  Genomics firm Nabsys has ... CEO, Barrett Bready , M.D., who returned to ... the original technical leadership team, including Chief Technology Officer, ... Product Development, Steve Nurnberg and Vice President of Software ... the company. Dr. Bready served as CEO ...
(Date:3/21/2016)... -- Unique technology combines v ...   Xura, Inc. (NASDAQ: ... communications services, today announced it is working alongside SpeechPro ... particularly those in the Financial Services Sector, the ability ... a mobile app, alongside, and in combination with, traditional ...
(Date:3/14/2016)... 14, 2016 NXTD ) ("NXT-ID" or ... market, announces the airing of a new series of commercials ... of March 21 st .  The commercials will air on ... Squawk on the Street show. --> NXTD ) ... mobile commerce market, announces the airing of a new series ...
Breaking Biology News(10 mins):