BIOTECH Project, University of Arizona
PCR of GFP in pBAD-gfpuv transformed E. coli
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Lab Investigation: Examining a Single Gene
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 attempt to replicate is called the Green Fluorescent Protein (GFP) gene. This gene codes for the GFP protein, a protein normally produced by jellyfish that you supposedly transformed into bacteria in a plasmid (pBAD-gfpuv). The protein can be excited by UV light. In the plates (LB agar + ampicillian) the bacteria transformed with the pBAD-gfpuv plasmid did not glow. Why do you think the bacteria did not glow?
Conducting PCR
Materials/Equipment Needed
For the class:
• Thermocycler
• Microcentrifuge (optional)
• Micropipet and sterile tips
• Bacteria colonies from LB agar + ampicillian
For each reaction add the following to a PCR thin walled tube:
• 20 µl 2X GoTaq (contains Taq polymerase, dNTPs, MgCl2, and buffer for ideal reaction and loading dye for electrophoresis).
• 10 µl forward primer
• 10 µl reverse primer
• a little dab of bacteria from one colony
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.
Some groups will amplify control reaction, for each control reaction add the following to a PCR thin walled tube:
• 10 µl each primer
• 20 µl 2X GoTaq
• 0.5 µl control DNA (GFP plasmid) for (+) control , orfor the (-) control add nothing to the reaction
Procedure
1. Label the PCR tube so that you can distinguish the samples in the tube.
2. Add 10µl primer of each primer to each tube. If necessary, gently tap you tube on the counter to get
all of the liquid to the bottom of the tube.
3. Add 20 µl GoTaq (green solution). Close the tubes and centrifuge briefly (10 seconds) to pool all of the liquid at the bottom of the tube (if you do not have a centrifuge then tap or shake the tube contents to the bottom of the tube).
4. Using the micropipet with a clean tip, just barely touch one of the colonies that you would like to
amplify the DNA to test for the presence of GFP. If you can see the bacteria on the tip you have
too much. Place the tip into the water in the PCR tube and pipet up and down once or twice to
dislodge the bacteria and mix. To the control PCR reaction add 0.5 µl of control DNA for + control
or nothing for - control.
5. Place your tube into the thermocycler to run the 'GFP' program. This program is
One cycle of:
• 94°C for 5 min (initial denaturation and E. coli cell disruption)
30 cycles of:
• 94°C for 30 seconds (denaturation of DNA)
• 55°C for 45 seconds (primer annealing)
• 72°C for 1.5 minutes (DNA extension)
One cycle of:
• 72°C for 7 minutes (Final extension)
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
• 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
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
You will load 20 µl of your PCR reactions and 20 µl of the 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. Each gel should have both positive and negative control samples.
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. Each gel should have one lane loaded with MW marker, load 20l.
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 100 V - 125 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. At this point you cannot see the DNA, what can you see and how do the different lanes compare?
11. Place your gel into the staining tray bring it to the staining station. Completely cover (submerge) the gel with methylene blue and cover the tray. Stain overnight.
Next day--Viewing the gel: Dispose of the methylene blue, and carefully place the gel onto a white light box. The gel is very fragile so take care to not break it. Draw a picture of your stained gel, be as accurate as possible in drawing the bands:
Analysis
• What do you see on your gel? Which bacteria samples were able to amplify the GFP gene from the pBAD-gfpuv plasmid? Why do you think you saw these results? What else could you do to ascertain the amplified DNA is the GFP gene? If the gene amplified is GFP why did your bacteria not glow? Come up with a hypothesis and how you would test it.
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