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:12/19/2018)... , ... December 19, 2018 , ... ... Science, announces the validated release of Limfinity® version 7.1. The new release enhances ... offering from RURO as 2018 comes to an end. , Validation protocols, and ...
(Date:12/14/2018)... ... December 14, 2018 , ... NOVA OCULUS PARTNERS, LLC, ... green light to immediately begin a clinical patient trial in Edmonton using their ... of Health Canada has granted what is called an Investigational Testing Authorization for ...
(Date:12/13/2018)... ... December 13, 2018 , ... Microbial Discovery ... ingredient. This has been part of their strategic plan and an important milestone ... creates custom fermentation opportunities for MDG within the $44 Billion human probiotic market. ...
(Date:12/5/2018)... ... ... STRmix™ – sophisticated forensic software capable of resolving mixed DNA profiles previously thought to ... of third-degree sexual assault, a felony punishable by imprisonment for up to 15 years. ... cushions on which the sexual assault was alleged to have taken place. , Jennifer ...
Breaking Biology Technology:
(Date:12/22/2018)... ... , ... On Wednesday, Dec. 12, Greenberg Traurig, LLP’s ... Chen, Greater China Managing Partner of L.E.K. Chen shared her insights on “Biopharma ... David J. Dykeman , co-chair of Greenberg Traurig’s global Life Sciences ...
(Date:12/19/2018)... ... ... NDA Partners Chairman Carl Peck, MD, announced today that Deb Koeneman, MS ... Radiological Health (CDRH), has been appointed a Partner in the firm. Ms. Koeneman has ... than 25 years of regulatory experience. In addition to her FDA career, she was ...
(Date:12/19/2018)... ... December 19, 2018 , ... OrthoSpace Ltd. (“OrthoSpace” ... Arthroscopy evaluating the use of novel surgical techniques, including the OrthoSpace InSpace™ ... a painful, disabling condition for which there are historically few treatment options. The ...
Breaking Biology News(10 mins):