12 PCR protocol
PCR basic protocol using GoTaq DNA polymerase
- In a sterile, nuclease-free microfuge tube, combine the following reagents on ice.
- You will prepare a master mix (you make enough for all your reactions in a single tube), aliquot the master mix into individual 0.2 mL PCR tubes, and then add the unique component to each tube (your DNA). Don’t forget to account for the volume of DNA you will use as you do your calculations.
- Include 10% extra in your master mix to account for loss (e.g, if you have three samples, make enough master mix for 3.3 samples)
The following protocol is used to prepare a 25 uL PCR reaction. Each time you set up a PCR, you will fill out the table and calculate how much of each reagent will go into an individual reaction and in your master mix. Don’t forget to make enough master mix for your CONTROLS. You should include a “No-template control” every time to make sure your reagents are not contaminated.
| Reagent | Final Concentration | Amount for a single 25 uL reaction | Total amount needed for all reactions |
| 5X Green GoTaq Buffer | 1X | ||
| 10 mM dNTPs | 200 µM | ||
| 25 mM MgCl2 | 1.5 mM (1.0–4.0mM*) | ||
| 10 µM mixture of Forward and Reverse Primers | 0.2 µM (0.05–1 µM*) | ||
| Taq DNA Polymerase (5 units/uL)
(Can dilute if necessary) |
0.625 units per 25 µl PCR | ||
| Nuclease-free water | to 25 µl final volume (don’t forget to include DNA volume) | ||
| To ADD AFTER aliquoting MM: | |||
| Template DNA | <250 ng per 25 uL PCR * |
*Indicates concentrations that may need to be optimized. You’ll put in the volume of DNA!
Notes on templates: You can use either plasmids or genomic DNA as your template. In general, use about 25-125 ng of genomic DNA for a 25 uL reaction, or about 10 ng of plasmid DNA.
When you are done setting up your PCR reactions, keep them on ice until you start the PCR machine.
PCR cycling conditions for GoTaq:
| Step | Temperature | Time | Number of cycles |
| Initial Denaturation | 95 °C | 2 min | 1 |
| Denaturation | 95 °C | 30 sec | Perform between 25-40 cycles |
| Annealing | 42–65 °C* | 30 sec | |
| Extension | 72 °C | 1 min per kb in amplicon | |
| Final extension | 72 °C | 5 min | 1 |
| Storage | 4 °C | Indefinite | 1 |
*To determine annealing temperature, calculate the melting temperature of each set of primers that you are using. You can do this here:
https://www.idtdna.com/calc/analyzer
You will use an annealing temperature 5°C LOWER than the lowest melting temperature of your primers. You may need to optimize the annealing temperature.
IMPORTANT: You must record the exact PCR cycling conditions that you used in your lab notebook. Don’t forget to include:
- The number of cycles you performed
- The annealing temperature
- The extension time