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.
-
Once you
have loaded
all the wells
with the
correct
dyes, it will
look like
this…
+
Lane #2
Lane #4
Orange G
Crystal
Violet
Lane #1
Lane #3
Lane #5
Malachite
Green
Bromophenol
Blue
Xylene
Cyanol
Also, each dye’s molecules have a
molecular weight….the heavier the
molecules, the slower they will move
across the gel and vise versa
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.
Questionable
Mixture goes
in the last lane
You will
have a
positive
electrode
at one end
and a
negative
at the
other…
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.
Next, write the purpose for this lab…..
Lab 6, Gel Electrophoresis Part A
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)
A
Malachite Green
329
B
Orange G
452
C
Bromophenol Blue
669
D
Crystal Violet
407
E
Xylene Cyanol
538
Charge
Travel
distance
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…..
Orange G
Malachite Green
-
Bromophenol Blue
Crystal Violet
Xylene 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