"

12 DNA AND TECHNOLOGY

Learning Objectives

  1. Complete protocol for gel electrophoresis..
  2. Load gel for agarose gel electrophoresis.
  3. Read results of agarose gel electrophoresis.

Activity

  1. What does it mean to amplify DNA?
  2. Which lab technique is used for amplification?
  3. Explain the relationship between the lab techniques of PCR and gel electrophoresis.
  4. Write down your answers to these questions and discuss with your group.

 DNA and Technology

All organisms store genetic information in the form of nucleotide sequences within deoxyribonucleic acid (DNA) and ribonucleic acids (RNA). The ordered sequence of A, C, G, and T (or U) nucleotides within DNA and RNA provide the instructions for how the individual pieces of cell machinery (i.e. proteins) are made. Proteins facilitate every living process from membrane transport and cell division to cellular respiration and photosynthesis. Therefore, understanding the structure, composition, and properties of DNA and RNA is essential for laboratory science in Biology. To that end, several technologies and techniques have emerged in the past century to allow us to manipulate, measure, and modify nucleic acids in a laboratory setting. You will learn about several of them in today’s lab.

Scenario

Nora loved eating out at fine dining restaurants almost as much as she loved her job at a biotechnology firm which analyzed DNA sequences for the purpose of DNA barcoding for species conservation. At her favorite local seafood restaurant, Jill’s Crab Shack, there is a new dish which features Fourchu Lobster. In the culinary scene it is known as the “Rolls Royce of Lobster.” Supposedly, the best lobsters in the world. Soon after it appears on the menu at the Crab Shack, Nora notices Fourchu Lobster on the menu at several other local restaurants — Blue Lobster, Fish Tales, The Salty Dive, and Taylor’s Seafood. Given the rarity of Fourchu, she feels suspicious and wonders if the restaurants are being honest?  Is something fishy going on?

As a foodie and a scientist, Nora decides to test it out. She has a colleague from Nova Scotia send her a tiny sample of DNA from a certified Fourchu Lobster. Then she works with the food distributors and head chefs at the local restaurants to obtain DNA samples of those lobsters as well. She uses PCR to amplify the DNA to ensure she has plenty of samples to repeat the test multiple times. Next, she runs each sample using gel electrophoresis.

Pre-Lab Resources

Review the following resources before coming to lab:

Materials and Methods

  • Agarose
  • Erlenmeyer flask
  • Graduated cylinder
  • Electrophoresis equipment + combs
  • 1x TAE buffer
  • Micropipets + Disposable tips
  • Samples (provided)

Procedure Gel Electrophoresis

1. Prepare an agarose gel by referencing the video above.

  • Gels may be prepared ahead of time
  • Mix 0.2 g of agarose with 40 mL of 1x TAE solution in a 250 mL flask
  • Cover flask with saran wrap and heat in microwave for one minute (agarose should boil) 
  • Use gloves to remove flask
  • SWIRL gently to be sure all of the agarose is dissolved
  • Place the flask on your table top. Be sure not to touch the hot glass! 
  • Wait approximately 10-15 minutes until it cools enough so it feels warm to the touch but will not burn (i.e., temperature of baby’s milk bottle)

2. Locate the casting stand. Place the gel tray in the casting stand and tighten the ends. Each casting stand can support two gels at the same time. Be sure to secure each gel tray tightly in the casting stand.

3. Insert one comb on the top of each tray close to either end. 

4. Slowly pour dissolved, cooled agarose into the tray. 

5. Rinse the flask immediately with warm tap water then rinse flask 3 times with distilled water.

6. Wait approximately 20-30 minutes until the gel sets (appears opaque). Be careful not to move gel while it is solidifying. 

7. Gently remove the comb and view the wells created by the comb. If the wells collapse, your gel is not set. 

Next, while you are waiting for your gel to solidify, practice using a micropipette.

This video shows you how Using the Pipette

Practice using a micropipette. Your instructor will demonstrate the proper use of the micropipette.

  1. While the gel is solidifying, practice using a micropipette to transfer 10 µL of practice loading dye. Remember, you are using very small amounts; 1 µL is equal to 1 millionth of a Liter.
  2. ALWAYS put a plastic tip on the micropipette. Push the pipette into the plastic tip.
  3. Hold the micropipette so your thumb is on top of the dispensing knob or plunger (shake hands with the pipette)
  4. ALWAYS keep micropipette vertically (up) when there is fluid in it. Never allow fluid to run back into the pipette by laying it down filled
  5. Use your thumb to control the speed of the plunger. ALWAYS release (lift your thumb up) SLOWLY.
  6. The micropipette has two stops. Press to the first stop. Push down harder to the second stop. SLOWLY release (lift your thumb up).
  7. Hold the micropipette in one hand vertically about eye level and bring your sample tube to the same level.
  8. Press to the first stop and do not release. Insert the pipette tip just under the surface of the solution. SLOWLY, release (let your thumb up).

Then, fill the sample wells

  1. Press to the first stop and continue to press to the second stop to fill the well with sample fluid. DO NOT RELEASE until you remove the tip from the well. SLOWLY release. If you do not keep plunger down until you remove the tip from the well, you will suck liquid back into the tip.
  2. Eject the used tip in a beaker.
  3. ALWAYS change tips for each new sample you need to pipette.
  4. Use the sample provided to practice loading a sample gel.

Finally, learn how to run the gel electrophoresis by watching this video:

Loading DNA Samples

  1. Carefully remove the gel tray from the casting stand and place the tray (with the gel on it) in the electrophoresis box. Be sure the wells are at the negative end. The DNA samples will run to the positive end.
  2. Measure 250 mL of USED buffer (1x TAE). Pour prepared running buffer solution (approximately 250 mL) into the gel box reservoirs to cover the gel. The gel should be completely covered about 1 mm in depth. Do not overload. Be sure no air is trapped underneath the gel tray.
  3. Press to the first stop. Insert the pipette tip just under the surface of the DNA sample. SLOWLY, release (let your thumb up).
  4. Fill your well by pressing down to the first stop. Then, continue to press to the second stop to release all the fluid. DO NOT RELEASE until you remove the tip from the well, then SLOWLY release.
  5. Load the samples – 20 µL into the gel in the order provided in the scenario.

Gel Electrophoresis

  1. Avoid shock by making sure the table top is dry. Do not turn the power on until it is dry
  2. Connect the top of the box with voltage cables. The negative cable is black and the positive cable is red. Be sure the wells are at the negative end
  3. Plug power supply into the outlet. Turn on Power. Turn voltage to 200 volts and run for approximately 15 minutes. Look through the apparatus to see migration. If the bands have not migrated sufficiently, continue to run for an additional 5 minutes.
  4. Turn off Power supply
  5. Disconnect leads from power supply
  6. Analyze gel

    Clean-up

    1. Return used buffer to the designated container
    2. After analyzing the gel, place the used gel in the designated container
    3. Wipe off the table top

Discussion

  1. While running a gel electrophoresis, do the larger fragments travel faster or slower than the smaller fragments?
  2. Does it matter if the wells are placed near the red electrode?
  3. If you collect a small sample of blood from a crime scene and you need to run many tests to identify the source of the blood, which would you perform first, gel electrophoresis or PCR

Assessment

  1. Are blood type tests preferred over PCR results for establishing parenthood?
  2. Why is PCR necessary for determining parenthood once we have a source of DNA? 
  3. Suppose you are working in a biomedical laboratory studying cystic fibrosis, which is caused by mutations to the human gene CFTR. You hypothesize that a new allelic variant of this gene has 1000 bp worth of DNA totally deleted out of the gene itself. How could you test your hypothesis (assume you have access to the full genomic DNA of a human with this variant allele).

Post-Lab Resources

The following resources may interest you after lab has been completed:

DNA and Technology Lab Source Material: Discussion and Scenario is by Professor Melissa Willis. Gel Electrophoresis Set Up is by Professor Mary Phillips. Introduction, Pre-Lab Resources, Assessment and Post-Lab Resources are by Professor Gary O’Meally. Licensed: CC BY NC SA

License

Icon for the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License

Biology 1414 Lab Manual Copyright © by Tulsa Community College is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License, except where otherwise noted.

Share This Book