Navigation Links
Embryonic stem cells, mouse

Multiporator Transfection Protocol Protocol No. 4308 915.039 12/1999 Cell line Embryonic stem cells (ES cells), mouse, R1. Passage 15 Transfection with Linearized targeting vector (12 kb) with neoR-selection marker Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium DMEM / 15% FCS, 2 mM glutamine, 1 mM pyruvate, 1 x MEM, 1 x nucleoside mix, 1 x Pen/Strep, 1000 U LIF Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Michael Bsl ZMNH Transgene Technologie Martinistr. 42 D-20246 Hamburg
Phone +49 40 42803 6663 Fax +49 40 42803 6659 e-mail:
  1. Harvest the cells 36-40 hours after the last passage, determine the number of cells and centrifuge the cells (5 minutes, 200 x g, at room temperature).
  2. Wash the cells in Hypoosmolar Electroporation Buffer: Resuspend the cells in 10 ml Hypoosmolar Buffer and collect them by centrifugation (for 5-10 minutes, 200 x g, at room temperature). Remove supernatant.

    Note: The overall incubation time in the Eppendorf Electroporation Buffer must not exceed 30 minutes to guarantee a successful electroporation!

  3. Resuspend the cells at a concentration of 1 x 107 cells/ml in Hypoosmolar Electroporation Buffer.
  4. Mix 750 l cell suspension with 50 l DNA solution (1 g/l in Isoosmolar Electroporation Buffer).
  5. Transfer 800 l cell/DNA solution into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 300 V Time constant (T) 100 s No. of pulses (n) 1
  7. After the pulse, let the cells shortly recover for 5 minutes at room temperature and then carefully transfer the cells to 10 ml ES-medium.
  8. Plate the cells onto 100 mm culture dishes prepared with inactivated mouse embryonic fibroblasts feeder layers at a density of 1-1.5 x 106 cells per plate.
  9. The next day change medium and start selection 24 hours after the electroporation with 250 g/ml medium of G418. Change selection medium every day.
  10. 7-8 days after the electroporation pick the surviving individual ES-cell colonies under a stereo microscope, culture and expand them individually. Take only colonies with sharp borders and proper morphology.
  11. After expansion freeze down at least one aliquot of each clone and use another for DNA preparation to identify correctly transfected clones originating from homologous recombination by PCR or Southern Blot analysis.

Detection methods for transfection:
Selection with G418 to identify G418 resistant, stably transfected colonies carrying the neoR marker of the linearized targeting vector.



Page: All 1 2 3

Related biology technology :

1. Primary cells, rat heart endothel
2. Primary cells, rat heart muscle
Post Your Comments:

(Date:10/11/2017)... N.C. (PRWEB) , ... October 11, 2017 , ... ... ARCS® Foundation President Andi Purple announced Dr. Suneel I. Sheikh, the co-founder, ... ( ASTER Labs ), Inc. has been selected for membership in ARCS ...
(Date:10/11/2017)... ... ... is a basic first aid supply for any work environment, but most personal eye wash ... if a dangerous substance enters both eyes? It’s one less decision, and likely quicker response ... piece. , “Whether its dirt and debris, or an acid or alkali, getting anything in ...
(Date:10/11/2017)... ... October 11, 2017 , ... Proscia Inc ., ... a Webinar titled, “Pathology is going digital. Is your lab ready?” with Dr. ... best practices and how Proscia improves lab economics and realizes an increase in ...
(Date:10/11/2017)... and LAGUNA HILLS, Calif. , Oct. ... Cancer Research, London (ICR) and University ... SKY92, SkylineDx,s prognostic tool to risk-stratify patients with multiple myeloma ... MUK nine . The University of Leeds ... partly funded by Myeloma UK, and ICR will perform the ...
Breaking Biology Technology:
(Date:8/15/2017)... LLC , a medical device company focused on improving the safety ... ISO 13485 Certification, the global standard for medical device quality management ... ... for the early detection of IV infiltrations. ... "This is an important milestone for ivWatch, as it validates our ...
(Date:6/23/2017)... ITHACA, N.Y. , June 23, 2017 ... a leader in dairy research, today announced a new ... help reduce the chances that the global milk supply ... this dairy project, Cornell University has become the newest ... Food Supply Chain, a food safety initiative that includes ...
(Date:5/16/2017)... TEANECK, N.J. , May 16, 2017  Veratad ... leading provider of online age and identity verification solutions, ... the K(NO)W Identity Conference 2017, May 15 thru May ... Ronald Regan Building and International Trade Center. ... across the globe and in today,s quickly evolving digital ...
Breaking Biology News(10 mins):