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:10/9/2017)... ... 2017 , ... At its national board meeting in North Carolina, ARCS® ... Departments of Physics and Astronomy, has been selected for membership in ARCS Alumni ... the 2015 Breakthrough Prize in Fundamental physics for the discovery of the accelerating expansion ...
(Date:10/9/2017)... San Antonio, Texas (PRWEB) , ... ... ... a new study published on October 5, 2017, in the medical journal, ... demonstrated equivalence with the gold standard, video EEG, in detecting generalized tonic-clonic ...
(Date:10/7/2017)... ... October 06, 2017 , ... ... genomic technologies, launched its ProxiMeta™ Hi-C metagenome deconvolution product, featuring the first ... accompanying cloud-based bioinformatics software to perform Hi-C metagenome deconvolution using their own ...
(Date:10/6/2017)... ... October 06, 2017 , ... The HealthTech Venture Network (HTVN) ... their fourth annual Conference where founders, investors, innovative practitioners and collaborators are invited ... competition showcasing early stage digital health and med tech companies. , This day-long ...
Breaking Biology Technology:
(Date:3/30/2017)... 30, 2017  On April 6-7, 2017, Sequencing.com will ... hackathon at Microsoft,s headquarters in ... focus on developing health and wellness apps that provide ... the Genome is the first hackathon for personal ... largest companies in the genomics, tech and health industries ...
(Date:3/28/2017)... 28, 2017 The report "Video ... Monitors, Servers, Storage Devices), Software (Video Analytics, VMS), and ... Global Forecast to 2022", published by MarketsandMarkets, the market ... is projected to reach USD 75.64 Billion by 2022, ... The base year considered for the study is 2016 ...
(Date:3/23/2017)... 2017 The report "Gesture Recognition and Touchless Sensing Market ... - Global Forecast to 2022", published by MarketsandMarkets, the market is expected to ... between 2017 and 2022. Continue Reading ... ... ...
Breaking Biology News(10 mins):