Eppendorf Multiporator - Electroporation Bedienungsanleitung

Multiporator Basis-Applikations-Anleitung · Basic Applications ManualElektroporation · Electroporation ® Titel_Rueck_d_e.fm Seite 1 Freitag, 9. Jun

34The minimum pulse voltage is dependent on the cell diameter following incubation in electroporation buffer as well as on the temperature and on the

35For most cell types, the 20- to 30-minute incubation period in hypoosmolar buffer, which is unavoidable due to the conditions of the experiment, ha

36b) Influence of nucleic acid purity Empirical studies have shown that EDTA and buffer salts such as HEPES or TRIS can drastically reduce transfectio

37A cell density of 1 x 10 6 cells/ml is recommended for electroporation since the electrical field is still effective on the cells in this range. Wh

38Cell-specific application protocols are available on the Eppendorf homepage at www.eppendorf.com. The list of applications is updated on a regular b

39 4.1.3.2 Testing the tolerance to hypoosmolar conditions The cells are incubated in hypoosmolar electroporation buffer for 30 minutes at room temp

40 Table 2 Minimum pulse voltages at which the cell membranes may be permeated, in relation to the diameter of the cell after incubation for 10 to15

414 Electroporation protocol4 Electroporation protocolElectroporation conditions must be optimized for every cell line for which no specific applicat

42If the transfection experiments do not turn out quite as expected, helpful information may be found in the following troubleshooting guide.The speci

43Gene product hasa toxic effect on the cell.The gene product itself or a high expression rate of the gene product may have a toxic effect on the cell

Basis-Applikations-Anleitung ...

44Pulse is too short. Extend the pulse length to increase permeability of the cell membrane, which can lead to a higher transfection rate. Note that t

45Cuvette has been usedseveral times.Using an electroporation cuvette several times may result in a non-homogeneous electrical field during electropora

46Order no:4308 000.015 Multiporator®for eukaryotics4308 000.023 Multiporator®for eukaryotics, bacteria and yeasts4308 000.031 Multiporator®for eukary

47Order no:940000505 Multiporator®for eukaryotics940000602 Multiporator®for eukaryotics, bacteria and yeasts940000700 Multiporator®for eukaryotics, ce

48Fig. 4a:Minimum pulse voltages at which the cell membrane is permeatedThe minimum pulse voltage is dependent on the cell diameter following 10- to 1

49Table 3The electroporation buffers supplied by Eppendorf are tested for the following important criteria: • Conductivity • pH value • Osmolarity •

50 8 Appendix 8 Appendix Multiporator ® Transfection ProtocolJurkat 1. Harvest the cells in the exponential growth phase and centrifuge them (5 t

Eppendorf Offices Eppendorf South Pacific Pty. Ltd.Tel. +61 2 98 89 50 00Fax +61 2 98 89 51 11E-mail: [email protected]: www.eppendorf.com.a

eppendorf ® is a registered trademark.B 4308 900.091-09/0308 · Printed in Germany Your local distributor: www.eppendorf.com/worldwide Eppendorf AG

27 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

28The phenomenon whereby a short, intensive current surge (pulse) is used to generate reversible openings (pores) in a membrane was first used in the

29The electroporation functions of the Multiporator ® are fundamentally different to those of other commercially available devices. The main differ

30 Release of aluminum during electroporation Commercially available disposable cuvettes usually contain aluminum electrodes. Aluminum is released f

31 Biophysical basics of the Multiporator's technique Crucial parameters for successful electroporation are the voltage and the length of the pu

32 Calculating the field strength The critical field strength which is necessary for electropermeation of the membrane can be calculated approximately.

33To ensure that maximum transfection rates are achieved, the electroporation parameters should be optimized for each new cell line. This section con