Setting Up PCR Reactions

The polymerase chain reaction, or PCR, is an incredibly useful and powerful technique for copying DNA. In Bio 6B, you'll use PCR in several different experiments, and there are several related pages:

Before you read this page, you should read the PCR background page, which explains what's in a PCR reaction and how it works. This page will tell you how to get it done in the lab.

Reactions and controls

You'll almost always do more than one PCR reaction. For example, in the PV92 and TAS PCR experiments, you'll compare DNA from each person in your lab group. We can call those the experimental reactions.  In addition, you'll need two kinds of controls every time you do PCR:

  • Negative control: A reaction tube with all the ingredients for PCR, but no DNA template (a little water is added instead of DNA). This tube should't produce a PCR product. If it does, it shows that one of your other ingredients was contaminated with DNA; this would mean that the results from your other tubes aren't reliable, because they might all be contaminated. This happens a lot in PCR.
  • Positive control: A reaction tube with all the ingredients for PCR and a reliable DNA template that has been used successfully in the past. This tube should produce a PCR product. This is important in case your other PCRs don't work. If positive control works and the experimental PCRs don't, it means that the problem is with the experimental templates. If none of the tubes, including the positive control, produces any PCR product, the problem is likely to be with the other ingredients or with the PCR machine.

All the experimental and control PCRs should contain exactly the same ingredients, with the exception of the template DNA. In order to be sure you are consistent in setting up the reactions it's important to premix most of the ingredients in a master mix, as described below.

Prepare a master mix

Here's the basic protocol for setting up a PCR experiment:

Diagram of master mix and reaction tube setup.

First, all the ingredients except the DNA templates are combined in a master mix (also called a cocktail). The master mix is pipetted into the individual PCR tubes, and finally a different DNA template is added to each tube. In this example there are four PCR tubes; that would normally include two experimental PCRs and a positive and a negative control.

Calculations

Before you can set up your PCR reactions, you will need to prepare the master mix that contains all the PCR ingredients except the template DNA. Figuring out the amounts to out into the cocktail will require a little calculation.

The table below shows all the ingredients you will need for PCR. All these components must be present in the proper concentrations to allow PCR to work. Each time you do a set of PCR reactions, you should make a table like this, and fill in the volumes as you calculate them.

 Ingredient Initial Concentration Volume For 1 Reaction Final Concentration Volume for (N+1) Reactions
Water  NA          μl  NA  
 Buffer  5x        μl  1x  
 MgCl2 25 mM        μl 1.5mM  
  dNTPs 10 mM        μl   200 μM  
 Foward Primer 10 μM         μl 1 μM  
 Reverse Primer 10 μM       μl  1 μM  
 Taq Polymerase  5 Units/μl      μl 1.25 Units   
DNA Template unknown 10 μl  NA Don't add to master mix!
Total:   50 μl   (N+1) x 40 μl = 

Initial concentration is the concentration of the stock solution. Final concentration is the concentration of that component in the PCR tube when it goes into the machine, after all the ingredients (including the template) have been added. In this example, the final volume of each PCR tube is 50 μl, including 10 μl of DNA template.

Calculate the amounts for one reaction

Even though you're going to make a master mix, start by calculating the appropriate amounts for one reaction. Each component must have the correct concentration in the PCR tube when it goes into the machine. For most of the components, you can calculate the starting amount using the dilution equation: C1V1 = C2V2 (see the Calculations page for more on how to do the math). Do the calculations in this order:

Buffer
It's called GoTaq reaction buffer, because it accompanies the GoTaq polymerase enzyme. Don’t confuse the enzyme with its buffer. The final concentration is "1x," which simply means the right concentration to support enzyme activity. The buffer's concentration can't be given in terms of molarity, because it's a mix of several ingredients, including ions required by the enzyme.
In this example, you would calculate the buffer like this: (5x)(starting volume in μl) = (1x)(50 μl). Solving for the starting volume would give you 10.0 μl.
MgCl2
This one is straighfforward: (25 mM)(starting volume in μl) = (1.5 mM)(50 μl). Solving for the starting volume gives 3.0 μl.
dNTPs
You need to do a unit conversion: (10 mM)(starting volume in μl) = (200 μM)(50 μl). I would convert the 200 μM final concentration to 0.2 mM. Answer 1.0 μl.
Primers
(10 μM)(starting volume in μl) = (1.0 μM)(50 μl). Answer:  5.0 μl for each of the two primers.
Taq polymerase
This one isn't a concentration, it's an amount. For enzymes "Units" means units of activity, or enough enzyme to catalyze a reaction at a certain rate. In this example, you need 1.25 Units of Taq per reaction. Solve it as a conversion: 1.25 Units(1 μl/5 Units) = 0.25 μl.
Water
Once you’ve figured out all the other ingredients, calculate how much water you’ll need to bring the volume up to 50 μl (including template). You need to calculate the amount of water last, but it's listed first in the table because you usually add it first because it's a large volume. It would be difficult to start out by pipetting a tiny amount into an empty tube.
For this example, the volume of water would be: 50 μl – (10.0 μl buffer + 3.0 μl MgCl2 + 1.0 μl dNTPs + 5.0 μl forward primer + 5.0 μl reverse primer + 0.25 μl Taq polymerase) – 10  μl template = 15.75 μl water. The total volume includes the template, even though it's not part of the master mix.

Now that you've figured out these amounts, don’t pipet each ingredient separately into each PCR reaction tube; instead, calculate the amounts for the master mix and then prepare that.

Calculate the amounts for the master mix

The master mix will contain everything but the template, so for each reaction you'll need 40 μl of master mix and 10 μl of template DNA. You'll want to make enough master mix for all your reactions. In principle, for the example shown here, you would be tempted to multiply the calculated (one tube) amounts by 4. However, if you do that, you'll probably run out of master mix, so it's always wise to make a little extra. In our lab, we just make enough master mix for one extra reaction. If you're doing 4 reactions (N = 4), you'd multiply your amounts by 5 (N+1). In this example, that would be 50 μl of buffer, etc.

Put these multiplied amounts in the right column; those are the amounts you'll pipet into your master mix. Notice that there's no template in the master mix; don't multiply the template amount by N+1, because each template is separate.

The total volume of cocktail in this case would be 40 μl x 5 = 200 μl. Make sure your individual amounts add up to the correct total. In this experiment, each tube will get 40 μl of cocktail and 10 μl of template DNA

Why we calculate it this way: some students are tempted to skip the one-tube calculations and directly start calculating the cocktail amounts. The calculatioin almost always ends up wrong, because the final volume for calculating concentraitons must include the template, but the template isn't part of the master mix.

Once you've done all the calculations, you need to think about how to properly handle all the PCR components. Read all the instructions below before making your cocktail!

Tips for setting up the reactions

It’s essential to follow some simple rules as you set up the reactions:

  • Keep everything cold. Keep all the ingredients (except the water), the master mix, and the PCR tubes tubes on ice.
  • Keep the Taq polymerase enzyme on ice. The enzyme should stay in its dedicated ice buckets throughout the lab; we'll pas around the bucket with enzyme. Only take the enzyme tubes off the ice for the time it takes you to pipet the enzyme. This shouldn’t take you more than 10 seconds.
  • Add the ingredients in the order listed in the table above. You’ll have to figure out the water last, but you should add it to the tube first. Add the enzyme last. The enzyme could be denatured if you add it before you add to an incorrect buffer solution, so it must go in last.
  • Never use a pipet tip twice –change tips every time. This prevents cross-contamination.
  • Keep all the ingredients in the bottom of their microfuge tubes, and mix each one with the pipet tip as you take it out. You need to see the tiny drop of liquid come out of the tip and mix with the ingredients already in the tube.
  • Make sure the master mix is thoroughly mixed before you pipet it into your PCR tubes.
  • Once you've prepared your master mix and pipetted it into the PCR tubes, stop. Don't add the DNA template until the instuctor tells you, because we're all going into the same PCR machine and need to start at the same time.

References & further reading

 How to: PCR Calculations and Quick Tips for PCR from Seeding Labs.

How to set up a PCR. Video from Synthetic Biology One.

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