Introduction to PCR Laboratory

The Basics

PCR (Polymerase Chain Reaction) does what it sounds like: it amplifies a target sequence within a DNA molecule. PCR is often nicknamed a “molecular xerox machine.” In order to perform PCR, you need a source of DNA, a set of primers, one for each strand of the DNA, to flank the region targeted for amplification, plus a DNA polymerase enzyme, the raw materials ATP, CTP, GTP, and TTP. PCR runs through 25-30 cycles of replication, and each cycle doubles the number of DNA copies made. A cycle has three components: Denaturation at high temperature (about 94^oC,separates double-stranded DNA into single strands); Annealing (50-65^oC to allow primers to hybridize/anneal to target DNA sites); replication (72^oC DNA polymerase binds and makes DNA). You can go to http://www.accessexcellence.org/AB/BC/PCR_Xeroxing_DNA.html and read more about PCR, and this page has links to graphics of PCR as well as a biography of the person who “discovered” PCR, Kary Mullis. See also the animation of PCR at the DNA Learning Center website: http://www.dnalc.org/resources/BiologyAnimationLibrary.htm.

In our experiment, we are targeting a 822 bp (basepair) sequence of Lambda DNA. Lambda is a bacteriophage (virus that infects bacteria) and we are using its DNA which is 48,502 bps long. Because the genome is relatively small, we can use manual cycling in two steps: boiling water bath for denaturing, 55^oC water bath for annealing. Replication occurs as the reaction warms up from annealing temperature to denaturing temperature. Take care not to go beyond the 20 seconds in boiling water because the enzyme can be destroyed at boiling.

Credits: This laboratory protocol owes much to the writeup by Bloom, Freyer and Micklos in “Laboratory DNA Science” but the new target site and new primers resulted from work done with the biotechnology program class of 2001.

We are using primers that target a 821 bp site from position 27219 to 28040 that includes a possible 493 B/H fragment that could be used in cloning experiment. Upstream primer sequence is 5’ATC GCC GAA CGA TTA GCT CT 3’; downstream primer is 5’ATC CGG CAC AGT ATC AAG GT 3’.

I. Amplification

Reagents: Supplies and Equipment:

PCR bead (contains polymerase, dNTPs, buffer)	micropipettors & tips
10 ml 0.05 ng/ml lambda DNA	Thermal Cycler
4 ml primer mix	disposable gloves
sterile distilled water

1. Put on disposable gloves.
2. Use a permanent marker to label your PCR tube containing the PCR bead.
3. To the bead in the tube, add 4 ml. of primer mix.
4. Next add the 10ml lambda DNA
5. Bring to final volume of 25 ml by add 11 ml of sterile distilled water.
6. Mix thoroughly.
7. Use a push pin & puncture the lid of your tube if using water bath (not needed for our thermal cyclers).
8. Write your initials on top of lid before proceeding.
9. Put your tube into foam floatie, and begin the cycling:

· 20 seconds in boiling water bath

· 1 minute in 55^oC water bath

· repeat total of 30 times!

One group could use the thermal cycler if you have one available, you would use the following parameters:

• 94^oC one minute
• 52^oC 45 seconds
• 72^oC one minute for 30 cycles.
  
  Place your tube into the thermal cycler . Make sure the 4 dummie tubes are placed in each of the 4 corners of the cycler. The thermal cycler is pre-progammed already. Follow the SOP (laminated copy inside box with thermal cycler) to select the “lambda” program. Don’t hit the “Yes” key for “run program” until ALL tubes are placed inside the thermal cycler.

(Did you notice the difference in the number of steps and timing on the water bath versus thermal cycler versions? In the water bath version, you can get by with two steps because the lambda genome is pretty small and the primers will be able to find the genome target quickly to anneal, and replication takes place as the temperature is going up or down between boiling and 55 degrees C.)

II. Electrophoresis of PCR Product.

Reagents Equipment and Supplies

PCR product – from above step	micropipettor and tips
100 bp Ladder(size markers)	beaker
loading dye	camera and film & transilluminator
agarose	electrophoresis chamber
1X TBE buffer	power supply
0.025% methylene blue stain	marker pen
	Combs & teeth for electrophoresis chamber

We run the PCR product on a gel to verify that you did get amplification and that it is the expected size piece (821 bps).

1. Preparing the Gel.

1. For this sample, we want to make a 1.2% gel.

2. Get beaker that is at least 2X to 4X greater volume than volume you are preparing – let’s make up 50 ml in 250 mL beaker.
3. Put 50 mL 1X TBE Buffer into beaker or flask.
4. Now measure out 0.6 g. agarose, and sprinkle SLOWLY into beaker, swirling or stirring to disperse evenly. It is best to let the agarose hydrate for 5-10 minutes in the container before heating so it will dissolve faster and better.
5. To dissolve completely, need TWO steps:

Using Microwave: Follow carefully – agarose can become superheated and boil over in microwave or when swirled. Wear insulating gloves and eye protection when working with heated agarose vessels.

6. Place plastic wrap over top and VENT – do NOT seal off beaker.
7. Heat till bubbles start to form – WATCH. STIR/swirl to mix every few seconds.
8. Now heat to boiling – stir/swirl – check that it is dissolved – use tongs or pot holder to prevent burning yourself. Do not overcook. Hold up to light to be sure agarose is dissolved.
9. Place on benchtop and cool till can be held in hand (about 55^oC.)

2. Casting the agarose gel.
1. Carefully pour agarose solution into casting tray to fill it to depth of about 1/3 of the comb teeth.
2. Allow gel to solidify – do not rush. Allow 20-30 minutes.
3. Place casting tray with gel in electrophoresis box. Wells should be closer to black end (positive pole) (RUN TO RED).
4. Pour 1X Tris/Borate/EDTA (TBE) buffer into electrophoresis box till the two ends of chamber are filled and the buffer just covers the surface of the gel. Do NOT overfill with buffer.

3. Load Gel and Electrophorese.
1. Collect your PCR tube along with a Ladder (use 100 bp Ladder if available).
2. Add 1-1.5 ml of loading dye into each tube.
3. Load 10-15 ml of each sample into separate wells, using a fresh tip for each sample.
4. Electrophorese at 125 volts until the bromophenol blue bands have moved 50-60 mm from the wells.
5. Turn off power supply, remove casting try from box, and transfer gel to weigh boat or shallow container for staining.
6. Buffer can be returned to stock and reused 2-3 times.

4. Staining the Gel, View, Photograph.
1. Put on disposable gloves during staining and cleanup.
2. Flood gel with 0.025% methylene blue solution and allow to stain for 20-30 minutes.
3. Use funnel to return methylene blue to staining stock – can be reused about 10 times.
4. Rinse gel in warm tap water, let it soak for several minutes – may take several changes and 1-2 hours before DNA bands become optimally visible.
5. Use Polaroid gel-documentation system: take Polaroid 667 color film, with aperture to f/8 and shutter on 1/125^th second. Press shutter once.
6. Place left hand firmly on top of camera to steady it. Firmly grasp small white tab and pull straight out from camera. A larger tab appears.
7. Grip large tab at its center, and in one steady motion, pull film straight out from camera. This initiates film development.
8. Allow film to develop for 45 seconds. Do not disturb.
9. After development time is over, separate print from negative by peeling back at the end nearest the large tab.
10. Wait to see results from first photo before making additional photos.

Results and Discussion

1. If your photo showed strong bands with your ladder lane but not the PCR lane, what could you conclude and how could this help a repeat experiment?

2. What would you expect to find if you substituted human DNA instead of Lambda DNA in this reaction?

        3.   Do you think you would see more or less PCR product using a thermal cycler and why?

4. What was the total quantity of lambda DNA in your PCR tube? ___________
5. What was the final concentration of lambda DNA in your PCR tube?_______