introduction to biotechnology - newbury park high...
TRANSCRIPT
Introduction to
Biotechnology
Taken from Textbook: Biotechnology Science for the New Millennium, Ellyn
Daugherty, 2012
Presentation includes
-micropipetting
-gel electrophoresis
What is Biotechnology?
A. Defined: the manipulation of living organisms or their components such as cells, tissues, or even organs to serve human needs.
1. Artificial Selection (Selective breeding) –
We will learn about these in the Genetics and
Evolution Units
a. Mendel’s Pea Plants
B. Humans have been manipulating
genes for a long time!
b. Darwin’s Pigeons
c. Dog Breeds (>100 dog breeds)
2. Brewers/Bakers
a. Production of alcohol
b. Production of breads (yeast
fermentation)
c. Production of cheeses
3. Today
a. Using cells to produce therapeutic
drugs (Insulin or EPO at Amgen)
b. Manipulating cells for
horticultural and agricultural uses
c. Pet industry (GloFISH)
C. What’s Cool?
1. During these labs you will be using the
same tools, techniques, and procedures
that are performed in Biotechnology
companies!
DNA Gel
PROTEIN
Gel
Gel Electrophoresis
Micropipetting
II. What is a micropipette?
A. In a normal chemistry class, solutions are
measured in large graduated cylinders with
volumes from 10 to 1,000 milliliters (mL).
B. In molecular biology, volumes are
measured in microliters (uL). (Very small:
1 L = 1 x10-6 L – millionths of a liter or
1000 L = 1mL)Milliliters
Microliters
How Do We Measure Small Volumes in Biotechnology?
micropipettes
C. Why use micropipettes?
1. The volume of liquid that you are working
with is very small so you need a precision
instrument.
2. Remember DNA is a “macromolecule” but it
is still very, very small and NOT easily seen
with the naked eye.
a. So a small volume of liquid can still hold a
lot of DNA!
D. Micropipette Use
1. The micropipette has 2 Stops:
a. The First stop is to take liquid
into the pipet tip
b. The Second stop is to dispense
the liquid into the receiving
container or gel well
Always Hold Micropipette and epitubes at eye level
III. Gel electrophoresis
A. Defined: Procedure used to separate and
analyze DNA fragments.
1. A mixture of DNA fragments are placed at
one end of a porous gel and an electrical
voltage is applied to the gel.
2. DNA has an overall negative charge because
of the phosphate group in the backbone
a. As a result, DNA will move in the gel when
a current is applied.
b. DNA will move toward the positive
electrode. “Opposites Attract”
DNA
Gel electrophoresis
DNA (Agarose) gel
- electrode + electrodeDNA fragments
buffer~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
b. How do the DNA fragments separate?
Gel electrophoresis
- electrode + electrode
current
buffer~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
~~~~~~~~~~~~~~~~~~~~~~~~
a. Large DNA fragments= move slower and
stay stuck toward the top of the gel (where you
loaded them)
b. Smaller DNA fragments = move faster and
farther away from the top of the gel
1. The pores in the gel restrict the movement
of the DNA fragments:
2. "Mice" run faster through the forest than
"elephants“:
a. Large DNA fragments are like the
“elephants”
b. Smaller DNA fragments are like the “mice”
C. Gel Loading Procedure
1. Make sure the dial on the micropipette
is set to desired volume
2. Add disposable pipet tip
3. Press plunger to first stop
4. Insert pipet tip into your solution
(just below the surface)
5. Slowly release plunger to bring
the liquid into the pipet tip
6. Move the pipet tip towards the gel well
to be loaded
a. Place tip ABOVE the well in the gel
(but under the buffer)
7. Press the plunger to the first stop to
transfer liquid into the well
8. Keep the plunger down as you remove
the tip from the well
Gel Loading Procedure
9. Eject tip into waste container
10. Write down which well you loaded on
the handout taped to the lab bench
Gel Loading Procedure
Micropipette tip should be
ABOVE the well NOT IN
IT!!!!
“Tripod Technique”
Micropipette tip punched right through the gel
See dye under the wells
Do NOT pierce through the bottom of the gel!
NICE!
Watch Video:
https://www.youtube.com/watch?v=uEy_NG
Dfo_8