Transcript Gel Electrophoresis

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
The negatively charged
particles move toward the
positive electrode while the the
positive charge particles move
toward the negative electrode.
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.
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.
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.