Polymerase Chain Reactions

What is PCR?

The videos linked below are a good general introduction to PCR.

PCR | Polymerase Chain Reactions

What is Polymerase Chain Reaction? | PCR Explained

PCR Components

DNA Template: The initial source of DNA material may be purified or small amounts of cells. It may be in various forms.
DNA Primers: Primers are pairs of short, single stranded DNA molecules that are used to prime the replication. You can custom select primers that search for specific genes or that replicate the whole sample.
Deoxynucleotide triphosphates (dNTPs): Raw nucleotides are needed, and dNTPs are the building blocks of DNA that the molecule will incorporate to the DNA chain.
DNA polymerase: This thermostable enzyme has the ability to incorporate raw nucleotides into the growing DNA chain. This enzyme is most often called Taq polymerase.
Buffer solution: This makes sure that the pH and other requirements for DNA polymerase are met.

PCR Variations

Standard PCR (PCR) amplifies a specific sequence of DNA with the goal of determining the identity of the isolate. Only yields qualititative data, ie the isolate is or is not a match for the primer.
Real time PCR (qPCR) uses the same PCR basics, but includes a probe that is used to track the progress of the PCR reaction. This method is most commonly used to quantify the amount of isolate in a sample when coupled with a DNA standard.
Droplet digital PCR (ddPCR) uses the same PCR basics and a probe, but encapsulates individual samples (whole cell bacteria, DNA, etc) in droplets, where the PCR reaction takes place. Droplets that contain the target bacteria will fluoresce and can be counted.

Standard PCR Materials

   10X Taq mutant buffer
    Forward and reverse primers
   100X OmniTaq polymerase
   200X dNTP's
   Sterile water
   DNA template (whole cells or extracted DNA)
   Thermocycler
   Thermocycler tubes (250uL capacity, sterile)
   Microcentrifuge tubes (1.5mL capacity, sterile)

Running a Standard PCR Trial

For a standard PCR, I like to use 20uL reactions with 5uL of DNA template. At this volume, a master mix is required because you cannot pipette the low volumes required. So, you must calculate your master mix. This mix will include: 10X buffer, forward and reverse primers, dNTP's, Taq polymerase, and sterile water. Here are the steps to produce a master mix and start a PCR reaction.

1. Determine the number of reactions you will be running. For each primer set you use, you must include a positive control (PC, DNA you know will react with the primer) and a no-template control (NTC, sterile water in the place of the DNA template). This means an additional 2 reactions! Make a note of this number.
2. Calculate the volume of each reagent needed for your number of reactions. This is done by generating a table like the one below. Generate a table for 1 reaction, and multiple each reagent voume by the number of reactions you plan to run (Step 1). If you are running less than 5 reactions, add one extra reaction. If you are running more than 5 reactions, add 3 extra reactions. Don't forget your two control reactions!

Reagent (stock concentration)	Concentration	Volume for 1 reaction	Example: Volume for 5 reactions
Buffer (10X)	1X	2uL	10uL
Forward primer (10uM)	500nM	1uL	5uL
Reverse primer (10uM)	500nM	1uL	5uL
dNTP (200X)	1X	0.1uL	0.5uL
Taq polymerase (100X)	1X	0.2uL	1uL
Sterile water	---	10.6uL	53uL
DNA template	---	5uL	---

3. Prepare and label a microcentrifuge tube for the master mix (all reagents except the DNA template). Combine the reagents into the tube using proper sterile technique. Vortex the tube well then briefly centrifuge on the table-top centrifuge.
4. Prepare and label a termocycler tube. It may be helpful to generate a code for each reaction as the tubes are small. Decant 15uL of the master mix into each thermocycler tube according to the number of reactions needed. Add 5uL of DNA template into each tube. For the control tubes add the following: control DNA into the PC and sterile water into the NTC. DO NOT add any DNA into the NTC tube!!! Centrifuge the thermocycler tube briefy.
5. Your samples are now ready to thermocycle. Place all tubes in the thermocycler and close the lid. Turn the thermocycler on and select "Protocol Library". Select the protocol titled "JELPCR". Select "Run Protocol". Change the volume to 20uL and select "Begin Run".
6. When the thermocycler is done running (held at 4degC), place the thermocycler tube in the fridge. You may prepare the gel while the PCR is running if you wish.