introduction to biotechnology - newbury park high...

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