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:2/9/2016)... ... February 09, 2016 , ... PharmApprove ... Policy for the National Organization for Rare Disorders (NORD). Dorman will lead PharmApprove ... voices are heard throughout the drug regulatory review process. , “Adding Diane Dorman ...
(Date:2/9/2016)... -- This market research report on the global microbiology ... the market in terms of revenue (USD Million). The ... manufacture of microbiology culture media and related products. The ... providing the overall information of various market segments included ... provides the overall information and data analysis of the ...
(Date:2/9/2016)... February 9, 2016 Three-Year Initiative Supports ... to Take Part in Life-Changing Camp ... designed to positively affect the lives of children born with rare ... --> SHPG ) is announcing a new initiative designed to ... well as the future of rare disease care. --> ...
(Date:2/9/2016)... , Feb. 9, 2016 ... Inhibitors-Pipeline Insights, 2016", report provides in depth ... development activities around the Protein-Tyrosine Phosphatase 1B ... product profiles in various stages of development ... II, Phase III and Preregistration. Report covers ...
Breaking Biology Technology:
(Date:1/20/2016)... 20, 2016  Synaptics Incorporated (NASDAQ: SYNA ... today announced sampling of S1423, its newest ClearPad ... small screen applications including smartwatches, fitness trackers, and ... and rectangular shapes, as well as thick and ... moisture on screen, while wearing gloves, and supports ...
(Date:1/15/2016)... , Jan. 15, 2016 Recent publicized ... small to find new ways to ensure data security ... iOS and Android that ties ... biometrics, transforming it into a hardware authorization token. Customer ... swipe their fingerprint on their KodeKey enabled device to ...
(Date:1/13/2016)... , January 13, 2016 /PRNewswire/ ... announced the addition of the  "India ... Estimation & Forecast (2015-2020)"  report ... http://www.researchandmarkets.com/research/7h6hnn/india_biometrics ) has announced the addition ... Identification Market - Estimation & Forecast ...
Breaking Biology News(10 mins):