Navigation Links

Multiporator Transfection Protocol Protocol No. 4308 915.028 11/1999 Cell line HL-60, human monocyte like cells (suspension cell line) Transfection with Plasmid pEGFP-N1 (in bidistilled H2O) Electroporation buffer Eppendorf Hypoosmolar Electroporation Buffer (PH) Culture medium RPMI 1640 / 10% FCS Cuvette Eppendorf, 4 mm gap width, 800 l Temperature RT (20-25 C) Reference Dr. Hans Hcker Institut fr Med. Mikrobiologie, Immunologie und Hygiene
Technische Universitt Mnchen Trogerstr. 4a D-81675 Mnchen
Phone +49 89 4140 4184 Fax +49 89 4140 4183 e-mail:
  1. Harvest the cells in the exponential growth phase and centrifuge them (for 5-10 minutes, 200 x g, at room temperature).
  2. Resuspend the cells in RPMI 1640 / 0.5% FCS, determine the number of cells and centrifuge them (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 in Hypoosmolar Electro poration Buffer. When doing so. set the cell concentration to 2.5 x 106 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) 760 V Time constant (T) 60 s No. of pulses (n) 1
  7. After the pulse, allow the cell suspension to stand in the cuvette for 5-10 minutes at room temperature.
  8. Carefully transfer the cell suspension from the cuvette to 3-5 ml RPMI 1640 / 10% FCS, and cultivate it in a 35 mm culture dish.
Detection methods for transfection:
The expression of the plasmid pEGFP-N1 can be detected clearly after 24-48 hours with the aid of FACS analysis or under a fluorescence microscope. Result: Survival rate: 80% Transfection rate: 44% based on the number of surviving cells
35% based on the initial number of cells used for the experiment Results were measured 24 hours after transfection.



Page: All 1 2

Related biology technology :

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

(Date:11/25/2015)... , Nov. 25, 2015  Neurocrine Biosciences, Inc. ... Gorman , President and CEO of Neurocrine Biosciences, will ... Conference in New York . ... visit the website approximately 5 minutes prior to the ... replay of the presentation will be available on the ...
(Date:11/25/2015)... ... 2015 , ... A long-standing partnership between the Academy of ... formalized with the signing of a Memorandum of Understanding. , AMA Executive Director ... Minter and Capt. Albert Glenn Tuesday, November 24, 2015, at AMA Headquarters in ...
(Date:11/25/2015)... 25, 2015 The Global ... a professional and in-depth study on the current ... (Logo: ) , The ... including definitions, classifications, applications and industry chain structure. ... international markets including development trends, competitive landscape analysis, ...
(Date:11/24/2015)... LUMPUR, Malaysia , Nov. 24, 2015 /PRNewswire/ ... global contract research organisation (CRO) market. The trend ... result in lower margins but higher volume share ... increased capacity and scale, however, margins in the ... Research Organisation (CRO) Market ( ), ...
Breaking Biology Technology:
(Date:11/20/2015)... 20, 2015 NXTD ) ("NXT-ID" ... the growing mobile commerce market and creator of the ... , was recently interviewed on The RedChip Money ... this weekend on Bloomberg Europe , Bloomberg Asia, ... --> NXTD ) ("NXT-ID" or the "Company"), a ...
(Date:11/17/2015)... , November 17, 2015 Paris ...   --> Paris from 17 th ... DERMALOG, the biometrics innovation leader, has invented the first combined ... on the same scanning surface. Until now two different scanners ... one scanner can capture both on the same surface. ...
(Date:11/12/2015)... , Nov. 12, 2015  A golden retriever that ... muscular dystrophy (DMD) has provided a new lead for ... Hospital, the Broad Institute of MIT and Harvard and ... . Cell, pinpoints a ... "escape" the disease,s effects. The Boston Children,s lab of ...
Breaking Biology News(10 mins):