ANALYTICAL TECHNIQUES IN BIOTECHNOLOGY

LABORATORY
(BT 1403)

R.ARUNA VIGNESHWARI
96507214001
ELECTROPORATION
What is electroporation?
 A short controlled pulse of electricity
to cell momentarily disrupting lipid
bilayer.
 Small pores (40-120nm) reseal
quickly.

Cell wall

Nucleus

DNA enters
PRINCIPLE
 Basic conditions for
electroporation
 Cell wall-less strain
 Transforming DNA
 Selectable markers
 Carrier DNA
INSTRUMENTATION
ELECTROPORATOR WITH CUVETTE
LOADED
Electroporator

25 microfarads = 2500 V
@ 200 ohms for 5 ms
PROCEDURE
Cell
incubation

Buffer containing foreign DNA

High voltage

Form pores in to cell

DNA enters
 Seems to cause
disruption in cell
membrane
 Reconstitution of
membrane leads to
large pores which allow
DNA molecules to enter
 Works for bacteria,
yeast and animal cells
Advantages

 Simple
 convenient
 Rapid
 Efficiency of transformation can be
improved
DISADVANTAGES
 · Cell Damage: If the pulses are of the
wrong length or intensity, some pores
may become too large or fail to close after
membrane discharge causing cell damage
or rupture .
 · Nonspecific Transport: The transport
of material into and out of the cell during
the time of electropermeability is
relatively nonspecific. This may result in
an ion imbalance that could later lead to
improper cell function and cell death
limitations
 Very low amt of dna inserted
 Regenertaion of plant is very easy
APPLICATIONS
Cell (electro)fusion
 Monoclonal
antibody production
 Cancer
immunotherapy
 Receptor insertion
into membrane
 Tissue regeneration
 Research (dissease
models)
 Electrochemotherapy =
= chemotherapy + electroporation
Electric pulse
generator
Chemotherapeutic Increased membrane Pores reseal
surrounds the cells permeability allows
access to the cytosol

Electrodes time
Tumour
Electric pulse application

Injecting chemotherapeutic
 Cancer Therapy Procedure Utilizing
EPT
Injection of
Bleomycin

Electroporation Therapy (EPT)

DC
Pulsing the Electroporation
Tumor with
applicator

Drug Surrounds Cell Poration, Cells Reseal

Tumor Cells drug enters cells And Die

Very effective treatment requiring low level of technical skills

THANK YOU