Name:

Date:

Period:

Lab Investigation: Examining a Single Gene Using PCR

BIG IDEA: In this lab investigation, you will learn about a technique called polymerase chain reaction (PCR) that allows us to examine a very small piece of DNA.

To look at a small piece of DNA, first you have to choose what DNA you want to look at, then you have to make a bunch of copies of that piece of DNA so you can see it.

• You will make copies of DNA is just like your cells do when they replicate DNA. What ingredients does your cell use when it replicates its DNA?



 

 
 
 
• Keeping in mind what a cell does when it replicates its DNA, make a list of steps involved in replicating DNA:
 





 
 
 
You will use some of these same ingredients and steps to replicate DNA in a test tube instead of a cell. The piece of DNA you will replicate is called the Green Fluorescent Protein (GFP) gene. This gene codes for the GFP protein, an protein normally produced by jellyfish that you transformed into bacteria in a plasmid (pGLO) The protein can be excited wUV light.

 
 Preparing yeast DNA

Materials/Equipment Needed

Procedure

1. Pipet 20 µl sterile water into the microfuge tube.
2. Use a sterile pipet tip on a pipetor to place a small amount of yeast into the sterile water in the tube, pipet the water/yeast mixutre up and down a few times to completely dislodge the yeast.
3. Close the tube and microwave for 2 minutes on high to burst the yeast and release their DNA.
4. The yeast DNA is now ready for PCR. Store in a freezer until ready to use.
 

 
Conducting PCR

Materials/Equipment Needed

For the class:
• Thermocycler
• Microcentrifuge (optional)
• Micropipet
• Micropipet tips

For each reaction:
• 0.2 ml microfuge tube
• 20 µl master mix [contains 10.75 µl sterile water, 1.25 µl MgCl2, 2.5 µl PCR buffer, 1.25 µl left-hand
primer (#1), 1.25 µl right-hand primer (#2), 2.5 µl nucleotides (also called dNTPs), 0.5 µl DNA
polymerase]
• 5 µl yeast DNA (wild-type or mutant)
 
Procedure

1. Label the microfuge tube.

2. Using the micropipet with a clean tip, add 20 µl master mix to your tube.

3. After adding the master mix, add 5 µl yeast DNA (either wild-type or mutant) to your tube. If necessary, gently tap your tube on the counter to get all of the liquid to the bottom of the tube.

4. Place your tube into the thermocycler to run the 'rad14' program. This program is 35 cycles of:

94°C for 45 seconds
55°C for 45 seconds
72°C for 3 minutes

5. After the cycles are complete, PCR reactions can be refrigerated or prepared for electrophoresis.
 

 
Electrophoresis of your PCR reactions

Materials/Equipment Needed

• Electrophoresis apparatuses, electrodes, and power supplies
• Micropipet
• Micropipet tips
• Loading dye
• 0.8% agarose gel
• Molecular weight markers (1 tube per gel)
• Water bath at 55°C or hot plate
• Thermometer for water bath
• TAE buffer
• Ethidium bromide paper (1 piece per gel)
• Staining tray
• Gloves (for handling ethidium bromide)
• UV light box
• UV light polaroid set up (including camera, film, camera connector, and light shield)
• Biohazard bag
 
Procedure

Pouring an agarose gel

1. Get your electrophoresis apparatus and seal both ends of the gel tray with tape or stoppers.

2. Make sure one comb is in place at the negative electrode (black end of the gel).

3. Pour melted agarose into the gel space until the gel is about 5 mm deep. Let the agarose
harden, which should take 5-10 minutes. Don’t touch/move your gel until it’s hard. In the meantime, prepare your PCR reactions for electrophoresis.

 
Electrophoresis of your PCR reactions

1. Using the micropipet with a clean tip, pipet 5 µl of gel loading dye into your PCR reaction tube.

You will load both your PCR reactions and standard DNA markers sample into the gel. A standard DNA marker has a bunch of different sized pieces of DNA so you can compare it to the DNA from your PCR reaction to figure out what size piece it is.

2. Draw a picture of your gel and label in which wells you will load which samples (PCR reaction(s), DNA marker).

3. When your gel has hardened, remove the tape or stoppers.

4. Load your samples into the wells - be sure you keep track of which samples you're loading in which wells.

5. Pour TAE buffer carefully so it fills the electrophoresis apparatus and just covers the gel.

6. Run that gel! Plug the electrodes into your electrophoresis apparatus (red to red, black to black),
being careful not to bump your gel too much.

7. Plug the power source into an outlet and set the voltage to about 100 V (max = 120 V).

8. Let the gel run until the dye migrates about 5-6 cm from the wells (about 20-25 minutes).

9. Turn off the power supply, disconnect the electrodes, and remove the top of the electrophoresis
apparatus.

10. Carefully remove the gel. The gel can be wrapped in plastic wrap and stored in the refrigerator or
placed in the staining tray for DNA staining.

 
Staining gels to examine PCR reactions

1. Place gel in staining tray

2. Using gloves, remove the plastic from the ethidium bromide sheet and place the ethidium bromide paper on the gel. Gently rub the paper with your fingers to make sure it is contacting the gel all over.

3. Stain for about 20 minutes.

4. Put the gel on the UV light box and, with the UV shield down, view your gel.

5. Take a polaroid picture of your gel.


Analysis

What do you see on your gel? Is the DNA copied from the wild-type yeast different from the DNA copied from the rad-14 mutant? Why or why not?

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona Last Modified March 12, 2002 Nadja Anderson, Ph.D. nadjal@email.arizona.edu http://biotech.biology.arizona.edu