Transcript Document

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 an obsicle to slow the
passage of molecules according to
their size and shape.
Restriction digest of DNA
 One of the basic tools of modern
biotechnology is DNA restriction enzyme
digestion.
 This is the process of cutting DNA into
smaller fragments using enzymes.
 This allows scientists to analyze and
compare DNA from different sources, and
to study the smaller more manageable
pieces of DNA.
 Because enzymes are very specific in their
effects on a substrate, they are
predictable and repeatable in their effects.
Restriction Enzymes
 The ability to cut DNA predictably is due
to the restriction enzymes specificity they
cut at only one sequence of nucleotides.
 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 their function of
restricting the growth of viruses through
the cutting of DNA with enzymes.
How does electrophoresis work?
• The gel is made from agar, a substance in seaweed.
•DNA has a negative charge on its particles.
• Molecules sort based on:
•Charge - The greater the charge the more pull.
•Size – Bigger pieces are slower, smaller are faster.
•Shape - Coiled is slower straight is faster.
•The negatively charged particles move toward the
positive electrode while the positive charge particles
Move toward the negative electrode. Agarose makes
them move slower and shorter distances if they are
big, and faster and further if they are small pieces
of DNA.
How does it work?
• DNA is cut into smaller fragments with
restriction enzymes that cut at only
certain sequences of nucleotides.
• Loading dye is used to indicate the
movement of the fragments of DNA.
• The negative DNA molecule is attracted
to the positive electrode.
•The smallest fragments move the
greatest distance.
Look Closely at the Gel and note
similarities and differences in bands
and patterns in the samples A - H
A
B
C
D
E
F
G
H
Analyzing a Gel
 Which lane contains the most fragments of
DNA?
 Which sample of DNA has the smallest and
largest fragments cut by the restriction
enzyme?
 Why do some bands of DNA fragments
appear in more than one lane of the gel?
 How could you use this technique to
analyze DNA?