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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 by UV light. In one group of plates (LB agar + ampicillian + arabinose) the bacteria transformed with the pGLO plasmid "glowed". In the other group of plates (LB agar + ampicillian) the bacteria did not glow. Why do you think the bacteria glowed or did not glow?

Since the same plasmid was added to both of the plates, you assume that the DNA is the same in the bacteria on both plates. Based on the above discussion, how could you determine if this is indeed correct?

 
 Preparing bacteria DNA

Materials/Equipment Needed

Procedure

1. Pipet 20 µl sterile water into the microfuge tube.
2. Use a sterile pipet tip on a pipet to place a small amount of bacteria into the sterile water in the tube, pipet the water/bacteria mixture up and down a few times to completely dislodge the cells.
3. Close the tube and microwave for 2 minutes on high to burst the cells and release their DNA.
4. The bacteria 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 add the following to a PCR thin walled tube:

· 5 µl extracted DNA

· 10 µl 2X GoTaq (contains Taq polymerase, dNTPs, MgCl 2 , and buffer for ideal reaction and loading dye for electrophoresis).

· 5 µl primer mix (includes both the forward and reverse primers)

These are small volumes; you will need to be sure to look at the pipette tip when you are pipetting to make certain that the components are being added.  

 
Procedure

1. Label the PCR tubes (DNA from the Amp only plates, or DNA from the Amp/Arabinose plates).

2. Using the micropipet with a clean tip, add 5 µl extracted DNA to the appropriate tube.

3. After adding the DNA, add 10µl GoTaq to the tubes. If necessary, gently tap your tube on the counter to get all of the liquid to the bottom of the tube.

4.   Add 5 µl of the primer mix to each tube.   Close the tubes and centrifuge briefly (10 seconds) to pool all of the liquid at the bottom of the tube.

5. Place your tube into the thermocycler to run the 'GFP' program. This program is 30 cycles of:

94°C for 30 seconds
55°C for 45 seconds
72°C for 1.5minutes

6. 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. Two or three groups can share a gel, but only one molecular weight marker is needed per gel.

2. Draw a picture of your gel and label in which wells you will load which samples (PCR reaction(s), DNA marker). Be certain to have the information of where the other groups added their samples.

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? Did the bacteria amplify the GFP gene from the pGLO plasmid? Why or why not? What else could you do to ascertain the amplified DNA is the GFP gene?

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