Download Electrophoresis Revised

Agarose Gel Electrophoresis
Grade Level: High School
Subject: Biology
Overview and Purpose:
The lab will allow the students to be a forensic detective in their own classroom by analyzing DNA they found
at the “crime scene” and comparing it to two suspects DNA. The students will be given a worksheet on Gel
Electrophoresis and will also be given picture or what the guilty person’s DNA looks like in Agarose Gel
Electrophoresis. They will use this picture to match one of the four suspected DNA that they have tested with
Agarose Gel Electrophoresis.
Objectives:
Learn about the properties and function of Agarose Gel Electrophoresis.
Background Information:
Electrophoresis is a method of separating substances based on the rate of movement while under the
influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is
created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and
then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab.
During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive
and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the
electrical current. Under an electrical field, DNA will move to the positive electrode (red) and away
from the negative electrode (black). Several factors influence how fast the DNA moves, including;
the strength of the electrical field, the concentration of agarose in the gel and most importantly, the
size of the DNA molecules. Smaller DNA molecules move through the agarose faster than larger
molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of
a dye that binds to DNA. -www.rochester.edu/College/BIO/labs/...files/GelElectrophoresis.doc
Print This For Students
Micropipettes used to accurately measure small volumes of liquids (volumes typically vary from 5 to
1000 μL)
Use of the micropipettes
Additional Notes:
Prepared By: Richard Kuhnke
Melissa Nolte
Illinois Learning Standards Addressed:
The standards that are addressed from goal 11 are:
11.A.4a, 11.A.4b, and 11.A.4c. These
standards include developing a hypothesis,
conducting a controlled experiment to test
the hypothesis and collecting, organizing,
and analyzing the data.
The standard that may apply from goal 12 is 12.A.4a,
it discusses the understanding of genetics.
Which is not wholly applicable but the
closest to the experiment.
There are no standards from goal 13 that I feel like
apply to this lesson.
Agarose Gel Electrophoresis
When you push down gently on the plunger of the micropipette, you will feel a “stop” where the
resistance increases. If you push a little harder, the plunger will move even further to a second stop.
The first stop is used to suck up the correct volume. The second stop is used to completely expel the
liquid you are measuring.
Liquid is never drawn into the barrel of the micropipette itself. An appropriate tip should always be
placed firmly on the end. Since the principle by which the micropipette works is the creation of a
vacuum in the tip, causing liquid to be drawn up, it is critical that the tip be on tight enough to make
an air-tight seal. Having said this, do NOT jam the tips on so hard that they are hard to get off. The
tips used for the 1000μL pipettes are usually blue.
The volume to be taken up is set by turning the plunger on the top of the micropipette and reading the
numerical settings displayed. A setting of 100 μL is equal to 0.100 mL. A setting of 1000 μL is
equal to 1.000 mL. Do not set the micropipette below 100 μL or above 1000 μL under any
circumstances! Doing this is essentially the only way that the micropipettes can be broken.
For the “suspects” we will use two different types of DNA. The students will be given a
worksheet and a picture or what the guilty person’s DNA looks like in Agarose Gel Electrophoresis.
They will use this picture to match one of the four suspected DNA that they have tested with
Agarose Gel Electrophoresis.http://wserver.scc.losrios.edu/~nussl/chem401/LABS/Lab%2001%20Pipetting.pdf
Loading the gel
• Add 6 l of 6X Sample Loading Buffer to each 25 l PCR reaction
• Record the order each sample will be loaded on the gel, including who prepared the sample, the
DNA template - what organism the DNA came from, controls and ladder.
• Carefully pipette 20 l of each sample/Sample Loading Buffer mixture into separate wells in the
gel.
• Pipette 10 l of the DNA ladder standard into at least one well of each row on the gel.
Note – if you are running multiple gels, avoid later confusion by loading the DNA ladder in different
lanes on each gel.
Additional Notes:
Agarose Gel Electrophoresis
Running the gel
• Place the lid on the gel box, connecting the electrodes.
• Connect the electrode wires to the power supply, making sure the positive (red) and negative (black)
are correctly connected. (Remember – “Run to Red”)
• Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on
the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes! .
• Check to make sure the current is running through the buffer by looking for bubbles forming on
each electrode.
• Check to make sure that the current is running in the correct direction by observing the movement
of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the
DNA).
• Let the power run until the blue dye approaches the end of the gel.
• Turn off the power.
• Disconnect the wires from the power supply.
• Remove the lid of the electrophoresis chamber.
• Using gloves, carefully remove the tray and gel.
Gel Staining
Additional Notes:
Agarose Gel Electrophoresis
• Using gloves, remove the gel from the casting tray and place into the staining dish.
• Add warmed (50-55°) staining mix.
• Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue).
• Pour off the stain (the stain can be saved for future use).
• Rinse the gel and staining tray with water to remove residual stain.
• Fill the tray with warm tap water (50-55°). Change the water several times as it turns blue.
Gradually the gel will become lighter, leaving only dark blue DNA bands. Destain completely
overnight for best results.
• View the gel against a white light box or bright surface.
• Record the data while the gel is fresh, very light bands may be difficult to see with time.
www.rochester.edu/College/BIO/labs/...files/GelElectrophoresis.doc
Micropipette use- http://wserver.scc.losrios.edu/~nussl/chem401/LABS/Lab%2001%20Pipetting.pdf
Making the Gels and Running Gel Electrophoresis
www.rochester.edu/College/BIO/labs/...files/GelElectrophoresis.doc
Animated Version- http://learn.genetics.utah.edu/content/labs/gel/
Set-Up Instructions:
Cast the gels the night before. For this lab you need to extract DNA previous to doing this experiment.
There is another stem kit in which this can be done
http://learn.genetics.utah.edu/content/labs/extraction/howto/. The teacher must also make sure
there are 2
Additional Notes:
Materials Included in Kit:
1.
Worksheets on DNA and what makes Gel
Electrophoresis work (preparing, loading, running
etc....)
2.
Story line for the crime committed with an
Agarose Gel Electrophoresis
different samples of DNA in order for the story line to connect with the lab.
Agarose Gel Electrophoresis Protocol
Preparing the agarose gel
• Measure 1.25 g Agarose powder and add it to a 500 ml flask
• Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size of the
casting tray)
• Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if using a
microwave, heat the solution for several short intervals - do not let the solution boil for long periods as
it may boil out of the flask).
• Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly.
• Seal the ends of the casting tray with two layers of tape.
• Place the combs in the gel casting tray.
• Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear
milky white).
• Carefully pull out the combs and remove the tape.
• Place the gel in the electrophoresis chamber.
• Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.
Note – gels can be made several days prior to use and sealed in plastic wrap (without combs). If the
gel becomes excessively dry, allow it to rehydrate in the buffer within the gel box for a few minutes
Additional Notes:
alibi for both subjects. With a picture of the guilty
persons DNA
3.
DNA (fish, horse, plant and human)
4. Buffer solution
5.
Micro-pipettes with extra tips
6.
Agarose Gel already prepared
7.
Electrophoresis Machine
8.
Ethidium bromide, silver, or Coomassie
Brilliant Blue dye to stain DNA
9.
Latex and Non-latex Gloves
10.
Overhead of the picture of guilty person’s D
Agarose Gel Electrophoresis
prior to loading samples.- www.rochester.edu/College/BIO/labs/...files/GelElectrophoresis.doc
Animated Version- http://learn.genetics.utah.edu/content/labs/gel/
Making the Gels and Running Gel Electrophoresis
www.rochester.edu/College/BIO/labs/...files/GelElectrophoresis.doc
Extraction Of DNA- http://learn.genetics.utah.edu/content/labs/extraction/howto/
Activity Timeline:
5 min- Background of the purpose and procedures Gel Electrophoresis along with a worsheet.
5-10 min- On the story line for the two suspects.
2-3 min On Using a Micropipette
5-10 min- Loading and Running Agrose Gel Electrophoresis
Depending- How long to run the Gels depends on Dye. It should be run until the Dye reaches the end
of the Gel.
2-3 min- Predictions of who is the Guilty person
20-30 Minutes for staining the Gels
Last couple of minutes analyzing the data gathered ( this could happen the next day also)
Safety Concerns: 1. Electricity
2. General lab safety
Additional Notes:
Additional Materials Needed:
1. Paper towels
2. Outlets
3. Space