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:8/9/2018)... ... August 08, 2018 , ... Since their establishment in 2012, ... with a team nearly double its’ size from last year, the company is thrilled ... turned heads and impressed crowds at a private product launch event during the ...
(Date:8/9/2018)... ... 09, 2018 , ... Simpson Gumpertz & Heger (SGH) will ... owners and managers for a full-day discussion on health care and life science ... for the first time on Thursday, 13 September 2018, at Harvard Medical School ...
(Date:8/1/2018)... ... August 01, 2018 , ... ... impact of a proprietary biofield energy treated supplement that potentially improves the ... for systemic and organ-specific inflammation, as well as, immune function response. The ...
(Date:7/31/2018)... ... July 31, 2018 , ... Mahoney Environmental , ... of their new website. The new website has been designed with improved functionality ... with Proceed Innovative to build a website created for the customer. The newly ...
Breaking Biology Technology:
(Date:8/15/2018)... ... August 15, 2018 , ... The Florida Chiropractic Association’s National Meeting ... Doctors from around the U.S. will gain firsthand insights into the new laser’s ... completed another successful clinical trial that has resulted in the granting of an ...
(Date:8/14/2018)... (PRWEB) , ... August 14, 2018 , ... ... and sleep monitoring solutions for the global scientific community, announced today the establishment ... of this new division is to foster and support scientific collaborations with ActiGraph’s ...
(Date:8/9/2018)... ... August 09, 2018 , ... ... gene therapy manufacturing facility has won Engineering News-Record (ENR) Texas & Louisiana’s 2018 ... for the Best Projects competition. The judges selected 42 projects across 18 categories ...
Breaking Biology News(10 mins):