7 anhang – Eppendorf Multiporator - Electroporation Benutzerhandbuch
Seite 25
25
7 Anhang
7 Anhang
Multiporator
®
Transfection Protocol
Jurkat
1. Harvest the cells in the exponential growth phase and centrifuge them
(5 to 10 minutes, 200 x g, room temperature).
2. Resuspend the cells in RPMI 1640 / 0.5 % FCS, determine the number of cells and wash them.
(5 to 10 minutes, 200 x g, room temperature).
Note
:
Incubation time in the 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 1 x 10
6
cells/ml.
4. Add and mix plasmid DNA (5 to 20 µg/ml final concentration, in bidistilled H
2
O).
5. Transfer 400 µl cell suspension into electroporation cuvettes (2-mm gap width).
The cell suspension must be free of air bubbles.
6. Electroporation:
Mode:
Eukaryotes "
"
Voltage (V)
240 V
Time constant (
τ
)
40 µs
No. of pulses (n)
1
7. After the pulse, allow the cell suspension to stand in the cuvette for 5 to 10 minutes at room temperature.
8. Carefully transfer the cell suspension from the cuvette to 3 to 5 ml RPMI 1640 / 10 % FCS and cultivate it in a
60-mm culture dish.
9.
Detection methods for transfection
:
The expression of the plasmid pEGFP-N1 can be detected clearly after 24 to 48 hours with the aid of FACS analysis
or under a fluorescence microscope.
Result
Survival rate:
70 to 85 %
Transfection rate:
65 to 80 % based
on the number of surviving cells
.
55 % based
on the initial number of cells used for the experiment
.
Results were measured 24 hours after transfection.
Cell line:
Jurkat, T-lymphocyte, human leukemia (suspension cell line)
Transfection with:
plasmid pEGFP-N1 (in bidistilled H
2
O)
Electroporation buffer:
Eppendorf hypoosmolar electroporation buffer (PH)
Culture medium:
RPMI 1640 / 10 % FCS
Cuvette:
Eppendorf, 2-mm gap width, 400 µl
Temperature:
RT (20 to 25 °C)
Reference:
Prof. Ulrich Zimmermann
phone:
+49 931 888 4508
Lehrstuhl für Biotechnologie
fax:
+49 931 888 4509
Biozentrum Universität Würzburg
e-mail:
Am Hubland, D-97074 Würzburg,
Germany
7.4 Protokoll für die Elektroporation von eukaryotischen Zellen am Beispiel Jurkat
Multipor_Appli_E_poration_de.fm Seite 25 Montag, 30. Januar 2006 2:16 14