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.



Page: All 1 2

Related biology technology :

1. Borrelia burgdorferi
Post Your Comments:

(Date:10/12/2017)... CA (PRWEB) , ... October ... ... ( ) has launched Rosalind™, the first-ever genomics analysis platform specifically designed ... bioinformatics complexity. Named in honor of pioneering researcher Rosalind Franklin, who made ...
(Date:10/11/2017)... ... ... wash is a basic first aid supply for any work environment, but most personal eye ... first if a dangerous substance enters both eyes? It’s one less decision, and likely quicker ... eye piece. , “Whether its dirt and debris, or an acid or alkali, getting anything ...
(Date:10/11/2017)... 2017  VMS BioMarketing, a leading provider of patient support ... Nurse Educator (CNE) network, which will launch this week. The ... health care professionals to enhance the patient care experience by ... other health care professionals to help women who have been ... ...
(Date:10/11/2017)... ... October 11, 2017 , ... A new ... rates in frozen and fresh in vitro fertilization (IVF) transfer cycles. ... to IVF success. , After comparing the results from the fresh and frozen ...
Breaking Biology Technology:
(Date:10/4/2017)... BLOOMINGTON, Ill. , Oct. 4, 2017  GCE Solutions, a ... powerful new data and document anonymization solution on October 4, 2017. ... the pharmaceutical field to comply with policy 0070 of the European ... documents and data. ... Innovation by GCE Solutions ...
(Date:6/23/2017)... and ITHACA, N.Y. , June 23, ... University, a leader in dairy research, today announced a ... to help reduce the chances that the global milk ... of this dairy project, Cornell University has become the ... the Food Supply Chain, a food safety initiative that ...
(Date:5/6/2017)... 2017 RAM Group , Singaporean ... breakthrough in biometric authentication based on a ... to perform biometric authentication. These new sensors are based ... by Ram Group and its partners. This sensor will ... chains and security. Ram Group is a next ...
Breaking Biology News(10 mins):