PCR for detecting phage

For this lab, you’ll use PCR to find out whether phage DNA is present in your bacterial cultures. If the virus is present, you should get DNA produced in your reaction and see a band on your electrophoresis gel. If no phage is present, you won’t get a band. In order to be sure the PCR is working, you’ll need a positive control and a negative control. The positive control will be the original phage stock, and the negative control will be a “plate lysate” from the “no phage control” plate – cells, but no phage.

Calculations for PCR

In the PV92 lab, the cocktail was premade for you, but this time, you’ll have to make it yourself. The cocktail will include all the PCR ingredients except the template DNA, which will be different for each reaction. Do all the calculations before you pick up the ingredients.

Here are the ingredients needed for PCR:

Water
Added to adjust the volume and concentration of the other ingredients. Must be pure and sterile.
Reaction buffer
Controls the pH and salt concentrations to allow the enzyme to do its job. The buffer is normally included in a separate tube when you buy the enzyme. It comes as a concentrate (usually 10x); you dilute it to make the buffer concentration 1x in the PCR reaction tube.
MgCl2
Mg2+ interacts with both nucleic acids (primers & template) and with individual nucleotides. If the concentration of Mg2+ is too low, amplification may not occur; if the concentration is too high, the wrong products might be produced. In many cases, MgCl2 concentration must be optimized experimentally for a specific reaction.
Nucleotides 
The monomers that will be used by the polymerase to make new DNA strands.
Primers
The oligonucleotides that give the polymerase a place to start. Must be present in high concentration. In this lab, the primers base pair with the PV92 region of your DNA.
Polymerase
The enzyme that makes the new DNA. Taq polymerase is commonly used in PCR.
Template DNA
The starting DNA, part of which will be copied in the PCR reaction.

All these components must be present in the proper concentrations to allow PCR to work. In order to ensure that all the reaction tubes get the same ingredients (aside from the DNA template), mix everything but the template in a cocktail. Make one cocktail with enough for all your reaction tubes, then pipet the cocktail into the individual reactin tubes, then add the appropriate template to each reaction tube.

How to do the calculations

Read all the instructions before making your cocktail!

  • First, calculate the appropriate amounts for one reaction. Once you’ve figured out all the other ingredients, calculate how much water you’ll need to bring the volume up to 50 μl.
  • You can use C1V1=C2V2 to figure out most of the amounts. The final volume for each reaction will be 50 μl, so V2 will be 50 μl. Keep in mind that this is the final volume in the PCR reaction tube, including cocktail and template. In this experiment, each tube will get 45 μl of cocktail and 5 μl of template DNA.
  • The buffer is called GoTaq reaction buffer, because it accompanies the enzyme. Don’t confuse the enzyme with its buffer.
  • Enzyme: The enzyme is called GoTaq DNA polymerase. The final amount of enzyme in each reaction should be 1.25 Units. That’s an amount, not a concentration; don’t use the C1V1=C2V2 formula.
  • Don’t pipet each ingredient separately into each PCR reaction tube; mix one cocktail large enough for all your reactions, then dispense the cocktail into each reaction tube, and finally add the DNA templates. In order to make the cocktail, multiply the numbers for one reaction by 5. You’ll actually have 4 PCR reactions, but you should always make a little extra cocktail (one extra reaction); otherwise, you’ll run out.
  • Don’t add template DNA to the cocktail; each reaction gets a different template.
  • Each reaction tube gets 45 μl cocktail; adding 5 μl template will bring the final volume up to 50 μl.
  • The ingredients are shown in the order in which you add them to the tube. You’ll have to figure out the water last, but you should add it to the tube first.

Use this table to figure out how much of each of these ingredients to use:

Ingredient [stock]  vol. for 1 reaction [final] vol. in cocktail for 5 reactions
 Water        
 GoTaq reaction buffer  5x    1x  
 MgCl2  25 mM    1.5 mM  
 Nucleotides  10 mM    200 μM  
 Primer T2F1  10 μM    1.0 μM  
 Primer T2R1  10 μM    1.0 μM  
 GoTaq DNA polymerase  5 Units/μl    1.25 Units  
Template DNA unknown 5 μl    don't add to cocktail
Total   50 μl    5 x 45 = 225 μl

 Setting up the reactions

Materials

Now you’re ready to start pipetting. Obtain the following materials:

  • Ice bucket with ice
  • Pipetmen, tips, and a beaker for waste tips
  • 1 500-µl micro tube for the cocktail.
  • 4 PCR tubes (Special tubes for PCR, not the usual micro tubes. They come in strips of 8 connected tubes; cut a strip in half, along with the lids.)
  • Sterile water
  • 5x GoTaq reaction buffer
  • 25 mM MgCl2
  • 10 mM Nucleotide mix (labeled nuc)
  • Primers T2F1 and T2R1, both 10 μM (labeled R and F)
  • GoTaq polymerase (As usual, you don't get the enzyme until you have added all the other ingredients to your cocktail tube.)
  • DNA templates:
  • Your 2 plate lysates: phage and no phage
  • T2 phage stock solution from the first lab (positive control; get this from the instructor)

Set up your Cocktail and PCR reaction tubes

Keep all the cocktail ingredients and your cocktail tube on ice. Before you pipet anything, make sure all the ingredients are thawed and mixed.

Label your cocktail tube and your PCR tubes (write on the sides, because the top may get smeared in the PCR machine; don’t use tape, because it might interfere with the tubes’ fit in the PCR machine).

Prepare your cocktail on ice, according to the table you filled in above. Be careful to pipet each ingredient into the bottom of the tube, and mix carefully with the pipet tip.

Pipet 45 µl cocktail into the reaction tubes. After adding 45 µl cocktail to each tube, you should have about 45 µl left in the cocktail tube. Don’t worry if it isn’t exact, but if you’re way off, you may have made a mistake. (How could you measure the leftovers in your cocktail tube?)

After setting up your cocktail, don't add the template DNAs to the tubes until the instructor tells you. We need to make sure all the groups are ready to go before you add the templates. The DNA shouldn’t be added until shortly before you start the PCR machine.

When the time comes, add 5 µl of the proper template to each reaction tube:

Tube # Template

  1. Phage lysate
  2. No-phage lysate
  3. T2 phage positive control DNA supplied by the instructor
  4. SM medium with no DNA template (negative control)

Once everyone has added their templates, put the reaction tubes in the PCR machine and start it. The machine will take more than an hour to go through all the cycles. After the PCR is complete, the DNA is fairly stable; it can sit at room temperature for a couple of days. Later, you’ll use electrophoresis to look at your PCR products.

The PCR Program

The PCR machine is already programmed to take your samples through the proper temperature steps. Each set of denature, anneal, and extend steps is called a cycle. The program for today’s reactions should be:

  1. Initial Denature 94° 1:00
  2. Denature 94° 1:00
  3. Anneal 54° 1:00
  4. Extend 72° 1:00
  5. Repeat steps 2-4: 30 cycles
  6. Final extension 72° 3:00
  7. Store 4° 00:00

Before you leave

  • If the PCR machine is still running, leave your PCR tubes in the machine.
  • Throw out the leftover cocktail along with your waste tips (biohazard).
  • Save all your DNA templates in the freezer in case the PCR doesn’t work.

Gel for phage PCR

The results for this experiment should be simple: each PCR sample should have a band if it contains phage DNA or no band if there is no phage DNA. The expected PCR products are only around 200 bp, so you should prepare a 2% agarose gel.

Load 25 µl of each sample, mixed with 2 µl sample buffer.

As a marker, load 10 µl of EZ Load Precision Molecular Mass Ruler (Bio-Rad) 100 bp ladder (this marker may already be mixed with sample buffer).

Since you're only trying to see the presence or absence of a band in each lane, you don't need to run this gel very far.

Review questions

  1. Which lanes on your gel are expected to have PCR products, and why?
  2. What makes this PCR specific for phage DNA?
  3. What is the positive control? Why are you using it? What will it mean if it gives you an unexpected result?
  4. What are the negative controls (there are two)? Why are you using them? What will it mean if a negative control gives you an unexpected result?
  5. What ingredients go into the PCR cocktail? What doesn't go into the cocktail?
  6. Why are you using a 2% agarose gel this time, when you used an 0.7% gel for the plasmid DNA in earlier labs?
  7. How do the cells get lysed in this experiment?
  8. Why don't you need to purify the phage DNA template for this experiment?
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