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Lab Investigation: Examining a Single Gene

In this lab investigation, you will use polymerase chain reaction (PCR) to examine a very small piece of DNA.

What proteins and   small molecules are involved in DNA replication inside a cell?

       1.

       2.

       3.

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.   

Which plate(s) contain transformed bacteria?  

How can PCR be used to confirm the presence of the GFP gene in the bacteria?

How can PCR be used to confirm the lack of the GFP gene in the bacteria?

What ingredients are needed for PCR?

      1.

      2.

      3.

      4.

Extracting the DNA

Materials/Equipment Needed

For the class:

· Microwave

· Micropipet

· Micropipet tips

For each bacterial strain:

· Toothpick

· Sterile water

· 1.5 ml microfuge tube

· Bacteria colonies from each LB agar + ampicillian

Procedure

1 Obtain two microfuge tubes with 20 µl sterile water already dispensed into them.    Label your tubes- glow/ no glow

2. One black stripe plate: 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. Three stripe plate: Repeat steps 1-2 with your second plate of bacteria in a second microfuge tube

4. Close the tubes and microwave for 2 minutes on high to burst the cells and release their DNA.

5. 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

· MasterMix   (from Eppendorf, you can use PCR beads or you can mix your own master mix, the one from Eppendorf is very economical).

For each reaction:

To a 0.2 ml PCR microfuge tube add

10 ml of Eppendorf 2.5X MasterMix which contains:

· sterile water

· MgCl 2

· PCR buffer

· nucleotides (also called dNTPs)

· Taq DNA polymerase

5 µl extracted DNA (DNA from glow and no glow bacteria)

5 µl of the forward primer

5 µl of the reverse primer

Procedure

1. Label the microfuge tube glow/ no glow

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

3. After adding the DNA, add 10µl master mix and 5 µl of each of the primers 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. 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 1.5 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 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 electrophoresisapparatus 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 electrophoresisapparatus.

10. Carefully remove the gel.   The gel can be wrapped in plastic wrap and stored in the refrigerator or placed it 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 10 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?