Navigation Links
Borrelia burgdorferi

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.506 12/2001 Microorganism Borrelia burgdorferi B31 Cell type Bacteria, gram negative Molecules injected Linear DNA (in water) Growth medium Barbour-Stoenner-Kelly (BSK) II medium without gelatin Washing solution PBS, without divalent cations; EPS (272 mM sucrose, 15% glycerol) Electroporation solution EPS (272 mM sucrose, 15% glycerol) Outgrowth medium BSK II medium Cuvette 2 mm gap width Reference Samuels D.S., et al 1994 Journal of Bacteriology 176 6045-6049

Making electrocompetent cells:

  1. Grow cells in 500 ml BSK II at 32 C to a cell density of 3-7 x 107 cells/ml.

  2. Harvest by centrifugation (4,000 x g, 20 min., 4 C). Wash twice in 60 ml cold PBS and pellet by centrifugation (3,000 x g, 10 min., 4 C).

  3. Wash three times in 20 ml cold EPS and pellet by centrifugation (2,000 x g, 10 min., 4 C).

  4. Resuspend the cells in 0.6 ml cold EPS.

Electroporation of c ells:

  1. Add 0.3 to 1 g DNA to 50 l of electrocompetent cells on ice. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.

  2. Wipe moisture from the cuvette and insert the cuvette into the device.

  3. Electroporation:


  4. Mode Prokaryotes O Voltage (V) 2,500 V Time constant (T) 5 ms
  5. Immediately add 1 ml BSK II medium, transfer cells to a 15 ml tube and add another 9 ml of BSK II. Incubate for 24-48 hours at 32 C with shaking.
  6. Plate 0.1 ml of culture on solid BSK II medium. Pellet the remaining 9.9 ml (4,000 x g, 10 min.), resuspend in 1 ml of supernatant and plate on selective plates. Incubate for 14 days, at 32 C in a humidified 5% CO2 chamber.

Expected results:

Transformation efficiency up to 103 transformants/g of DNA.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. Borrelia burgdorferi
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:4/27/2016)... ... April 27, 2016 , ... The Board ... the appointment of John Tilton as Chief Commercial Officer.  Mr. Tilton joined Biohaven ... the founding commercial leaders responsible for the commercialization of multiple orphan drug indications. ...
(Date:4/27/2016)... MIAMI (PRWEB) , ... April 27, 2016 , ... ... a mutual endorsement of an Asia-Pacific Symposium as other research and development initiatives for ... of Santiago officials and top Global Stem Cells Group executives began meeting to establish ...
(Date:4/26/2016)... ... ... The European Patent Office (EPO) today announced that U.S. Biophysicist ... Inventor Award 2016 in the category "Non-European countries." The winners of the 11th edition ... on June 9th. , The human capacity to walk with fluidity is the sum ...
(Date:4/26/2016)... ... April 26, 2016 , ... Seattle based non-profit, The ... from 1Plus12 Corporation. The grant will be used to further the scientific research ... organization's website http://www.ivsci.org , In accounting the grant to the IVS, ...
Breaking Biology Technology:
(Date:3/14/2016)... NXTD ) ("NXT-ID" or the "Company"), a company ... of a new series of commercials on Time Warner Cable ... .  The commercials will air on Bloomberg TV, Fox Business ... show. --> NXTD ) ("NXT-ID" or the "Company"), ... the airing of a new series of commercials on Time ...
(Date:3/10/2016)... , March 10, 2016 ... new market research report "Identity and Access Management Market ... Audit, Compliance, and Governance), by Organization Size, by Deployment, ... 2020", published by MarketsandMarkets, The market is estimated to ... 12.78 Billion by 2020, at a Compound Annual Growth ...
(Date:3/9/2016)... GARDENS, Fla. , March 9, 2016 /PRNewswire/ ... management authentication and enrollment solutions, today announced the ... DigitalPersona ® Altus multi-factor authentication platform. ... and InfoSec managers to step-up security where it,s ... Washington, DC . ...
Breaking Biology News(10 mins):