A Process to DYE for

Lab 6: Molecular Biology

Part A

In these two labs (Part A and B) we will be using a separation technology called Gel electrophoresis.

Gel- we use a gelatin Agarose

Electro- using electricity

Phoresis- means ‘to carry across’

You are a lab technician mixing up a batch of stain (mixture of various dyes) for one of the researchers in your

lab.

You had started making a large batch when you were interrupted during the procedure.

When you returned to the stain, you could not remember if you had added all of the components (geez- pay attention to

your job, loser)…..

Rather than waste the whole batch of stain and start over, you will employ gel electrophoresis to determine if all of the dyes

are present in the mixture.

You will load your gel into the electrophoresis container.

Next, you will cover your gel with an electrolyte buffer…this is a liquid that will conduct electricity.

Using a micro-pipette, you will load 10 micro-liters (µL) of each dye into a different well in the gel.

Questionable Mixture goes

in the last lane

Lane #5

Xylene Cyanol

Lane #4

Crystal Violet

Lane #3

Bromophenol Blue

Lane #2

Orange G

Lane #1

Malachite Green

Once you have loaded all the wells

with the correct

dyes, it will look like this…

You will have a positive

electrode at one end

and a negative at

the other…

-

+

When you turn the electricity on, the molecules in the dye will begin to move across the gel….if

the molecules have a negative charge, they will move toward the positive and vise versa.

Also, each dye’s molecules have a molecular weight….the heavier the

molecules, the slower they will move across the gel and vise versa

What about the well with the questionable mixture?

1. Since it’s mixed with dyes of different weights, some will move faster than others….

2. Also some of the dyes in the mixture might be negative, others positive…

So they will separate from each other and you’ll be able to see which dyes were put into the mixture…..

So first, let’s hypothesize over what you think will happen with each dye when the electricity is turned on

First make a Table of Contents entry and write the title of this lab on the page you are using for Part A.

Lab 6, Gel Electrophoresis Part A

Next, write the purpose for this lab…..

Purpose: to understand the principles and practice of gel electrophoresis. Also, to demonstrate separation of molecules based on charge and size.

Underneath Purpose, write Dye Hypothesis: and create the following table:

Dye Names Molecular Weight (MW)

Charge Travel distance

A Malachite Green 329

B Orange G 452

C Bromophenol Blue 669

D Crystal Violet 407

E Xylene Cyanol 538

Using the pictures of the dye’s molecules, hypothesize if you think the charge of the molecule is neg. or pos. AND determine in order which dyes

you think will travel farthest to shortest distance.

THEN after your filled in table….write Pre Lab Question: and write answers with complete sentences to the pre lab question.

AND- Create your Data Collection table to fill in tomorrow…..

Malachite Green Orange G

-

Bromophenol Blue

Crystal VioletXylene Cyanol

Pre Lab Question:

1. Write a brief function for each of the parts used in gel electrophoresis:

a. Agarose gel

b. Electrophoresis buffer

c. Wells in the gel

d. Electric current

Data:Dye Name Dye Well # Migration Distance (mm) Migration Direction (+/-) Dye Molecules “Speed”

Rankings