Navigation Links
Optimizing electroporation parameters

Other important parameters for optimizing electroporation.
Adjustment of the electroporation buffer A precondition for successful electroporation with the Multiporator is the special buffer system with low electrical conductivity.

The best possible transfection results are obtained by using the original buffers from Eppendorf, which have been tested for sterility as well as for the absence of mycoplasma, endotoxins and pyrogens, and which have also under-gone a thorough cytotoxicity test.

Users may also make up this buffer themselves (see Table 1).

< TD vAlign=top align=middle>Eppendorf Isoosmolar Electroporation Buffer (PI) (no.: 4308 070.510)
Eppendorf Hypoosmolar Electroporation Buffer (PH) (no.: 4308 070.501)
Sterile bidistilled water Fill up to 1000 ml Fill up to 1000 ml
KCl 25 mM 25 mM
KH2PO4 0.3 mM 0.3 mM
K2HPO4 0.85 mM 0.85 mM
myo-Inositol* ad 90 mOsmol/kg ad 280 mOsmol/kg
pH value 7.2 0.1 7.2 0.1
Conductivity at 25C 3.5 mS/cm 10% 3.5 mS/cm 10%
Shelf-life 3 years 3 years
Table 1: Eppendorf Electroporation Buffer components

*The purity of myo-Inositol may vary greatly from batch to batch. It must be pure enough to ensure that, at 280 mOsmol/kg in bidistilled water, a conductivity of 10 S/cm is not exceeded. The conductivity of individual myo-Inositol batches should be measured before the preparation of the buffer.

Ideally, electroporation should be carried out in hypoosmolar buffer, in which the cell absorbs water shortly before the pulse and then swells up as a result. A number of effects, including a decreased optimal pulse voltage ensure that the plasma membrane can be permeated more easily.

For most cell types, the 2030-minute incubation period in hypoosmolar buffer has no effect on the viability of the cells. However, the incubation in hypoosmolar buffer may induce apoptosis, or even lysis, in very sensitive cells. Therefore, it is strongly recommended to test the tolerance of the cells to hypoosmolar conditions. The easiest way of doing so is by incubating the cells for 30 minutes in hypoosmolar buffer and then performing a viability stain using trypan blue or propidium iodide. If observation under a microscope reveals lysis in more than 10% of the cells, the osmolarity of the buffer must be increased by adding isoosmolar buffer. To determine the optimal osmolarity, it is advisable to incubate the cells in different mixing ratios of hypo- and isoosmolar buffer for 30 minutes prior to the experiment (see Table 2).

This 30-minute period is the maximum incubation time for the cells in the electroporation buffer system. A new viability test followed by observation under a microscope determines the osmolarity that can be tolerated by the cells. The mixing concentrations can then be used for all subsequent experiments with this cell type.

Irrespective of the buffer system selected, it is essential to ensure that the cells do not remain in the electroporation buffer for longer than 30 minutes.

It is recommended to transfer the cells 510 minutes after the pulse into growth medium and incubate them for at least 23 hours at 37C before any centrifugation takes place. This step markedly increases the survival rate.

Desired osmolarity Eppendorf Hypoosmolar Buffer (ml) Eppendorf Isoosmolar Buffer (ml)
90 mOsmol/kg 10.0 0.0
150 mOsmol/kg 6.8 3.2
200 mOsmol/kg 4.2 5.8
250 mOsmol/kg 1.6 8.4
280 mOsmol/kg 0.0 10.0
Table 2.< /b> Volumes of Eppendorf Hypoosmolar and Isoosmolar Electroporation Buffers to be used to adjust the desired osmolarity (final volume: 10 ml).



Page: All 1 2 3 4 5

Related biology technology :

1. Optimizing Transfection Conditions for Studying Signal Transduction Pathways
2. Optimizing pfuturbo DNA Polymerase Amplification Reactions with Perfect Match PCR Enhancer
3. Optimizing electroporation parameters
4. Optimizing Büchi® Rotary Evaporator Applications
5. Optimizing electroporation parameters
6. Optimizing Büchi® Rotary Evaporator Applications
7. Optimizing Bchi Rotary Evaporator Applications
8. Optimizing electroporation parameters
9. Optimizing electroporation parameters
10. Optimizing electroporation parameters
11. Optimizing electroporation parameters
Post Your Comments:

(Date:11/24/2015)... 2015 Cepheid (NASDAQ: CPHD ) today ... following conference, and invited investors to participate via webcast. ...      Tuesday, December 1, 2015 at 11.00 a.m. Eastern Time ...      Tuesday, December 1, 2015 at 11.00 a.m. Eastern Time ... New York, NY      Tuesday, December 1, 2015 ...
(Date:11/24/2015)... (PRWEB) , ... November 24, 2015 , ... ... maintain healthy metabolism. But unless it is bound to proteins, copper is also ... Health (NIH), researchers at Worcester Polytechnic Institute (WPI) will conduct a systematic study ...
(Date:11/24/2015)... Inc., a worldwide provider of clinical research services headquartered in ... has set a new quarterly earnings record in Q3 of 2015.  ... Q3 of 2014 to Q3 of 2015.   ... the establishment of an Asia-Pacific office to ... and Mexico , with the establishment ...
(Date:11/24/2015)... ... ... The Academy of Model Aeronautics (AMA), led by its Executive Council, has ... Prix, to represent the First–Person View (FPV) racing community. , FPV racing has exploded ... of racing and several new model aviation pilots have joined the community because of ...
Breaking Biology Technology:
(Date:10/29/2015)... YORK , Oct. 29, 2015 ... technology, announced a partnership with 2XU, a global ... to deliver a smart hat with advanced bio-sensing ... and other athletes to monitor key biometrics to ... the strategic partnership, the two companies will bring together ...
(Date:10/27/2015)... 2015 Synaptics Inc. (NASDAQ: SYNA ), the ... has adopted the Synaptics ® ClearPad ® ... its newest flagship smartphones, the Nexus 5X by LG ... --> --> Synaptics works closely ... collaboration in the joint development of next generation technologies. ...
(Date:10/26/2015)... -- Delta ID Inc., a company focused on bringing secure ... announced its ActiveIRIS® technology powers the iris recognition feature ... NTT DOCOMO, INC in Japan . ... include iris recognition technology, after a very successful introduction ... 2015, world,s first smartphone to have this capability. ...
Breaking Biology News(10 mins):