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:10/16/2018)... ... October 16, 2018 , ... ... first Congressional briefing on a revolutionary technology that effectively treats pain, reduces the ... Infinity Healing, testified on how technology using photobiomodulation (PBM) has been effective for ...
(Date:10/11/2018)... (PRWEB) , ... October 10, 2018 , ... ... the biotech community, announces the appointment of Wendy Nelson, PhD, as its new ... operational and R&D experience in the biotech industry. Most recently, Dr. Nelson ...
(Date:10/5/2018)... (PRWEB) , ... October 04, 2018 , ... ... diagnostics with genomics, along with Duke University, announced today at IDWeek™ 2018 the ... test that detects pathogen DNA in plasma, accurately identified causative pathogens in patients ...
(Date:10/2/2018)... BASEL, Switzerland (PRWEB) , ... ... ... leading provider of advanced software solutions for biopharmaceutical R&D, today announced a ... ambr® mini-bioreactor systems from Sartorius, a leading international pharmaceutical and laboratory equipment ...
Breaking Biology Technology:
(Date:10/11/2018)... ... October 09, 2018 , ... ... and supports high-growth, high-impact life science and biotechnology companies, announced today that ... disease foundations to discuss their investment philosophies, explain their fund and deal ...
(Date:10/11/2018)... ... October 11, 2018 , ... The American College of ... Patricia Flatley Brennan, RN, PhD, Director of the National Library of Medicine (NLM), during ... AMIA’s Annual Symposium is taking place Nov. 3 – 7. , In honor of ...
(Date:10/5/2018)... ... October 04, 2018 , ... ... University of Illinois Extension and Monroe County Economic Development Corporation (EDC), has ... Institute of Food and Agriculture (NIFA) Non-Land Grant College of Agriculture (NLGCA) ...
Breaking Biology News(10 mins):