Transcript electrophoresis_power_point

Gel Electrophoresis
What is Gel Electrophoresis?
Gel electrophoresis separates
molecules on the basis of their
charge and size. The charged
macromolecules migrate across a
span of gel because they are
placed in an electrical field. The
gel acts as a sieve to to retard the
passage of molecules according to
their size and shape.
BIOTECHNOLOGY
 One of the basic tools of modern biotechnology
is gene splicing.
 This is the process of removing a functional
DNA fragment ( a gene) from one organism
and combining it with the DNA of another
organism to study how the gene works.
 The desired result is to have the new organisms
carry out the expression of the gene that has
been inserted.
Restriction Enzymes
 The ability to cut and paste DNA
predictably is due to the use of restriction
enzymes.
 They were first identified in and isolated
from the bacteria that use them as a natural
defense mechanism to cut up the invading
DNA of bacteriophages – viruses that infect
bacteria.
 They are named for the
Gel Electrophoresis
 Electrophoresis is the movement of molecules
by an electric current.
 Nucleic acid moves from a negative to a
positive pole.
Gel Electrophoresis
 When DNA is applied to a macromolecular cage or gel
such as agarose or polyacrylamide, its migration under the
pull of the current is impeded.
Gel Electrophoresis
 Slab gel electrophoresis can have either a
horizontal or vertical format.
 Sample is introduced into wells at the top of
the gel.
Very Large DNA Molecules are Separated by Pulsed
Field Gel Electrophoresis (PFGE).
The movement of molecules is impeded in the gel so
that molecules will collect or form a band according to
their speed of migration.
% agarose:
2%
4%
5%
500 bp
500 bp
200 bp
200 bp
500 bp
50 bp
200 bp
50 bp
50 bp
The concentration of gel/buffer will affect the
resolution of fragments of different size ranges.
How does electrophoresis work?
• The gel is made from agar
• DNA is a negative molecules
• Molecules sort based on
•Charge
•Size
•shape
What is agar?
Agar comes from sea weed.
What is it used for?
The gel is 1% agarous and has
no electrical charge.
Electrophoresis Buffers
 Carry current and protect samples during
electrophoresis.
 Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE),
Tris Phosphate EDTA (TPE) used most often for
DNA.
 10 mM sodium phosphate or MOPS buffer used for
RNA.
 Buffer additives modify sample molecules.
– Formamide, urea (denaturing agents)
How does it work?
• DNA is cut into smaller fragments.
• Loading dye is used to indicate the
fragments of DNA are behind the dye
• The negative DNA molecule is
attracted to the positive electrode.
•The smallest fragments move the
greatest distance.
Electrophoresis Equipment
Horizontal or submarine gel
Electrophoresis Equipment
Combs are used to put wells in the cast gel for
sample loading.
– Regular comb: wells separated by an “ear” of gel
– Houndstooth comb: wells immediately adjacent
Running a Gel
 Use the proper gel concentration for sample
size range.
– 0.5–5% agarose
– 3.5–20% polyacrylamide
 Use the proper comb (well) and gel size.
Running a Gel
Load sample mixed with tracking dye (dye
+ density agent).
Running a Gel
 Detect bands by staining during or after
electrophoresis
 Ethidium bromide: for double-stranded DNA
 SyBr green or SyBr gold: for single- or
double-stranded DNA or for RNA
 Silver stain: more sensitive for single- or
double-stranded DNA or for RNA and
proteins
Procedure
 Remove comb and observe wells.
 Place carbon paper in each end of the tray.
 Cover with buffer, making sure the allow buffer to
overflow into each end of the tray.
 Load gels.
 Connect the electrodes.
 Turn on power supply.
 Allow gels to run – make sure you see bubbles
coming from the electrodes.
PROCEDURE (CONTINUED)
 It will take about 30 minutes for the gel to
run.
 Turn off power supply and remove
electrodes.
 Pour off buffer into the designated
container.
 Carefully remove gel from gel box and
place in glad container and cover with stain.
 Store in appropriate location.
What is significant about the
bubbles?
 They indicate that electrolysis of water is
taking place.
 One electrode will have a lot of bubbles and
the other will have a lesser amount. Why
the difference?
 The formula for water is H2O and the
splitting of the molecule will produce twice
as many atoms of hydrogen.
Summary
 Electrophoresis is used to separate molecules by size
and/or charge.
 Nucleic acid fragments can be resolved on agarose
of polyacrylamide gels.
 PFGE is used to resolve very large DNA fragments.
 CGE is more rapid and automated than slab gel
electrophoresis.
 The choice of electrophoresis method depends on
the type and size of sample.