Navigation Links
721.221

Multiporator Transfection Protocol Protocol No. 4308 915.050 11/2001 Cell line 721.221, EBV transfected B cell line Transfection with pcDNA3.1/V5-His-TOPO (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI / 10% FCS (200 U/ml penicillin; 200 g/ml streptomycin, 2 mM glutamine) Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Britta Eiz-Vesper Medizinische Hochschule Hannover Institut fr Transfusionsmedizin
Feodor-Lynen-Str. 21 D-30625 Hannover
Phone +49 160-3680653 Fax +49 511-5322079 e-mail: eiz-vesper.britta@mh-hannover.de
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 300 x g, at room temperature).
  2. Resuspend the cells in RPMI / 0.5% FCS, determine the number of cells and centrifuge them (for 5-10 minutes, 300 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 in Hypoosmolar Electroporation Buffer. When doing so, set the cell concentration to 2 x 107 cells/ml.
  4. Add and mix plasmid DNA (25 g/ml final concentration, in bidistilled H2O).
  5. Transfer 800 l cell suspension into electroporation cuvettes (4 mm gap width). The cell suspension must be free of air bubbles.
  6. Electroporation:

    Mode Eukaryotes Voltage (V) 400 V Time constant (T) 50 s No. of pulses (n) 2
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5 minutes at room temperature.
  8. Carefully transfer the cell suspension with a pasteur pipette from the cuvette into 5 ml RPMI / 10% FCS, and cultivate them in a little culture flask.
Detection methods for transfection:
Selection and limiting dilution assay for stable transfection after 48 hours. Result: Survival rate: 85% Transfection rate: 40% based on the number of surviving cells. Results were measured 48 hours after transfection.


'"/>

Source:


Page: All 1 2

Related biology technology :

1. 721.221
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:10/11/2017)... Tampa Bay, Florida (PRWEB) , ... October 11, ... ... Food and Drug Administration (FDA) has granted orphan drug designation to SBT-100, its ... antibody (sdAb) for the treatment of osteosarcoma. SBT-100 is able to cross the ...
(Date:10/10/2017)... ... October 10, 2017 , ... ... program has won a US2020 STEM Mentoring Award. Representatives of the FirstHand program ... in Volunteer Experience from US2020. , US2020’s mission is to change the trajectory ...
(Date:10/10/2017)... Calif. , Oct. 10, 2017 SomaGenics ... from the NIH to develop RealSeq®-SC (Single Cell), expected ... for profiling small RNAs (including microRNAs) from single cells ... Program highlights the need to accelerate development of approaches ... "New techniques for measuring levels ...
(Date:10/9/2017)... Oct. 9, 2017  BioTech Holdings announced today ... which its ProCell stem cell therapy prevents limb ... The Company, demonstrated that treatment with ProCell resulted ... saved as compared to standard bone marrow stem ... resulted in reduction of therapeutic effect.  ...
Breaking Biology Technology:
(Date:4/19/2017)... , April 19, 2017 The ... vendor landscape is marked by the presence of several ... however held by five major players - 3M Cogent, ... companies accounted for nearly 61% of the global military ... companies in the global military biometrics market boast global ...
(Date:4/13/2017)... , April 13, 2017 UBM,s Advanced ... will feature emerging and evolving technology through ... Innovation Summits will run alongside the expo portion of ... sessions, panels and demonstrations focused on trending topics within ... advanced design and manufacturing event will take place June ...
(Date:4/11/2017)... Apr. 11, 2017 Research and Markets has ... report to their offering. ... The global eye tracking market to grow at a CAGR of ... Eye Tracking Market 2017-2021, has been prepared based on an in-depth ... market landscape and its growth prospects over the coming years. The ...
Breaking Biology News(10 mins):