Gel Electrophoresis

1
Gel Electrophoresis
2
Gel Electrophoresis

• Uses
• DNA Fingerprinting!
• Diagnosis of Genetic Disease
• Paternity testing
• Separating or isolating proteins
• Separating or isolating specific DNA sequences

3
Gel electrophoresis
GEL
Power source

• Gel Electrophoresis uses electricity to separate
  molecules based on
• Size
• Charge

-
BUFFER
4
Separation of molecules

• Material to be separated is loaded into the wells
  of the gel
• Separated by Charge
• Molecules with a positive charge will move _____
• Molecules with a negative charge will move ______

Wells
-

THE GEL
5
Separation of molecules

• Separated by size
• Large size molecules will move ________
• Small size molecules will move _________
• What can you tell me about the molecules
  separated in this gel?

-

6
Making a DNA fingerprint

• Overall DNA is a negatively charged molecule.
• Which way will it move?
• What would happen if we put your DNA in the gel?

-
7
Making a DNA Fingerprint

• In order to make a unique pattern for an
  individual DNA must be cut into pieces
• Restriction enzymes are used to cut DNA at
  specific sites

8
Restriction enzymes

• Isolated from bacteria
• Originally function to break down foreign (viral)
  DNA
• Also used in making recombinant DNA

9
How do restriction enzymes make a DNA fingerprint?

• Before putting DNA into gel, cut it with
  restriction enzyme.
• These pieces of DNA are called RFLPs-restriction
  fragment length polymorphisms

-

• Each individual makes a unique pattern--a
  fingerprint

10
Guilty??
11
Real DNA fingerprinting

• Is a little more complicated
• Non-coding repeating sections are cut using
  restriction enzymes to make RFLPs (restriction
  length polymorphisms)
• These must be specifically detected from all of
  your genome using probescalled southern blotting

12
Our Lab

• We will be cutting the DNA from a virus called
  lambda (?)
• Lambda is a virus that normally infects E. coli
• We will cut the DNA with 3 different restriction
  enzymes
• BamHI
• EcoRI
• HindIII

13
The gel

• We will then load the samples into the gel and
  separate them using electrophoresis
• What do you expect to see?

14
Prelab Questions
15
PCR

• Very small amounts of DNA can be amplified using
  PCR
• PCR Polymerase Chain reaction
• Quick way of copying a lot of DNA

16

• Add your own
• Primer
• DNA polymerase
• Heat/Cool cycles
• Lots of DNA!!