> For the complete documentation index, see [llms.txt](https://salis-lab.gitbook.io/protocol-book/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://salis-lab.gitbook.io/protocol-book/build-genetic-systems/dna/pcr.md).

# PCR

A method for amplifying DNA *in vitro*. PCR is a revolutionary method developed by Kary Mullis in the 1980s. PCR is based on using the ability of DNA polymerase to synthesize a new strand of DNA complementary to the offered template strand. Because DNA polymerase can add a nucleotide only onto a preexisting 3'-OH group, it needs a primer to which it can add the first nucleotide. This requirement makes it possible to delineate a specific region of template sequence that the researcher wants to amplify. At the end of the PCR reaction, the specific sequence will be accumulated in billions of copies (amplicons).

## Overview

The steps of PCR visualized in **Figure 1** are as follows: 1. **Denature**, or melt, the DNA to allow separation of your double stranded DNA template into single stranded DNA fragments. 2. **Anneal** the primers to the template DNA. 3. **Extension** of the DNA polymerase to "copy" the template. 4. Repeat steps many times.

[![](/files/-LEjG9DkB2vuX9hqxsLl)](https://github.com/)

**Figure 1**. The basic steps of PCR.

Cases when you will use PCR:

* To amplify DNA that you have only a small amount of (e.g. a `gblock`, `PCR assembly product`, etc.)
* To amplify a DNA sequence off of a genome or a plasmid, known as colony PCR.
* To amplify DNA fragments for any of the possible cloning methods (e.g. `Digest-Ligate`, `Gibson Assembly`, `LCR`, etc.)

What you'll be doing for PCR: 1. Design a pair of primers for each amplicon (region of DNA to amplify) 2. Prepare template 3. Prepare PCR mix 4. Run PCR [thermal cycler](https://github.com/reisalex/salis-lab-protocol-book/tree/453898c9360786eef221e6fffd8409c03a547e50/13_Lab_Equipment/thermal_cycler.md) protocol 5. Analyze by [gel electrophoresis](https://github.com/reisalex/salis-lab-protocol-book/tree/453898c9360786eef221e6fffd8409c03a547e50/10_Test/gel_electro.md)

## Primer Design

Primers, as illustrated in **Figure 1**, are relatively short ssDNA oligos that will direct DNA polymerase during the PCR. There are two primers for a PCR reaction. The first primer, also known as the `Forward Primer` (FP), should hybridize to the 5'- end (left) of the `template strand` (the bottom strand in **Figure 1**). This means that the forward primer should be the same sequence as the `coding strand` (top strand in **Figure 1**). The second primer, also known as the `Reverse Primer` (RP), should hybridize to the `coding strand` on the 3'- end (right). This means that the reverse primer *must be* the reverse complement of the `coding strand`.

When you design primers, you *must* follow these rules if you want your PCR to work correctly:

1. Primers should be of similar length. Target 18-30 bp in length for the hybridization region. Specifically, the annealing temperature of the primers works best around 57-60°C. See the tools listed below.
2. "GC clamps" on the 3'- end of the primers. Aim for 2-3 bp of "GC" base pairings on your primer end.
3. Avoid strong DNA structures with ΔG < -5.0 kcal/mol.
4. Avoid self-dimers, or primers that bind to themselves with ΔG < -5.0 kcal/mol.
5. Avoid hetero-dimers, or pairs of primers that bind to each other with ΔG < -5.0 kcal/mol.
6. Primers should not bind to other locations with up to \~5 mismatches.

Tools that we use are listed here. Make sure to use Nearest Neighbor energy models for the melting temperature and structure predictions, as they are most accurate:

* [OligoAnalyzer](https://www.idtdna.com/calc/analyzer)
* [OligoCalc](http://biotools.nubic.northwestern.edu/OligoCalc.html)
* Biopython has a [MeltingTemp Module](http://biopython.org/DIST/docs/api/Bio.SeqUtils.MeltingTemp-module.html) if you want to do things in Python.

Online primer design guidelines and tools can be found [here](https://openwetware.org/wiki/Designing_primers).

You can add 5' tails on your primers. This will introduce extra DNA on the ends of your amplicon. Up to 15-20 bp tails won't really affect the PCR efficiency. Reasons for doing this might be to introduce [restriction enzyme](/protocol-book/build-genetic-systems/restriction-cloning/restriction-enzyme-digestion.md) cut sites to aid in your cloning. NOTE: The annealing temperature is still only calculated by the hybridization of the subsequence of the primer that binds to the DNA template, *not* the whole primer sequence.

## Choosing the Right DNA Polymerase for YOUR PCR

Refer to [NEB's DNA Polymerase Selection Chart](https://www.neb.com/tools-and-resources/selection-charts/dna-polymerase-selection-chart) and if it suits you, watch their [video](https://www.neb.com/tools-and-resources/video-library/choose-the-right-dna-polymerase-for-pcr). We have used the following DNA polymerases

## Q5 DNA polymerase

[Q5® High-Fidelity DNA Polymerase](https://www.neb.com/products/m0491-q5-high-fidelity-dna-polymerase#Product%20Information) has 2-fold higher fidelity than its predecessor, [Phusion DNA polymerase](/protocol-book/build-genetic-systems/dna/pcr.md#-Phusion-DNA-polymerase). FOr longer (>1 kb) amplicons, a Q5 PCR with fewer (25) cyclers should be considerd, especially if point mutations have been a problem for this amplicon.

Combine the following reagents together in a PCR tube. Add ddH2O first and Q5 DNA polymerase last. Transfer to the thermocycler and run the protocol as describe below.

| COMPONENT                         | 25 µL REACTION | 50 µL REACTION | FINAL CONCENTRATION |
| --------------------------------- | -------------- | -------------- | ------------------- |
| 5X Q5 Reaction Buffer             | 5 µL           | 10 µL          | 1X                  |
| 10 mM dNTPs                       | 0.5 µL         | 1 µL           | 200 uM              |
| 10 uM Forward Primer              | 1.25 µL        | 2.5 µL         | 0.5 uM              |
| 10 uM Reverse Primer              | 1.25 µL        | 2.5 µL         | 0.5 uM              |
| Template DNA                      | variable       | variable       | 0.01 - 10 ng        |
| Q5 High-Fidelity DNA Polymerase   | 0.25 µL        | 0.5 µL         | 0.02 U/µL           |
| 5X Q5 High GC Enhancer (optional) | (5 µL)         | (10 µL)        | (1X)                |
| ddH2O                             | to 25 µL       | to 50 µL       |                     |

| STEP                 | TEMPERATURE (°C) | TIME (s)     |
| -------------------- | ---------------- | ------------ |
| Initial Denaturation | 98°C             | 30 sec       |
| (1) Denature         | 98°C             | 5-10 sec     |
| (2) Anneal           | 50-72°C (Tm+3)   | 10-30 sec    |
| (3) Extend           | 72°C             | 20-30 sec/kb |
| REPEAT (1-3)         | 35 times         | N/A          |
| Final Extension      | 72°C             | 2 min        |
| Hold                 | 4°C              | Forever      |

[NEB Protocol](https://www.neb.com/protocols/2013/12/13/pcr-using-q5-high-fidelity-dna-polymerase-m0491)

## Phusion DNA polymerase

A legacy polymerase that we sometimes still order. No reason to use this over [Q5 DNA polymerase](/protocol-book/build-genetic-systems/dna/pcr.md#-Q5-DNA-polymerase). The [NEB Protocol](https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530) describes reaction setup and thermal cycler conditions for Phusion.

## Taq DNA polymerase

A cheaper thermo-stable polymerase relative to Q5 and Phusion. Used for small amplicons, [PCR Assembly](/protocol-book/build-genetic-systems/dna/pcr-assembly.md), and colony PCR off the genome.

Combine the following reagents together in a PCR tube. Add ddH2O first and Q5 DNA polymerase last. Transfer to the thermocycler and run the protocol as describe below.

| COMPONENT                     | 25 µL REACTION | 50 µL REACTION | FINAL CONCENTRATION  |
| ----------------------------- | -------------- | -------------- | -------------------- |
| 10X ThermoPol Reaction Buffer | 2.5 µL         | 5 µL           | 1X                   |
| 10 mM dNTPs                   | 0.5 µL         | 1 µL           | 200 uM               |
| 10 uM Forward Primer          | 0.5 µL         | 1 µL           | 0.2 uM               |
| 10 uM Reverse Primer          | 0.5 µL         | 1 µL           | 0.2 uM               |
| Template DNA                  | variable       | variable       | <1,000 ng            |
| *Taq* DNA Polymerase          | 0.125 µL       | 0.25 µL        | 1.25 units/50 µL PCR |
| ddH2O                         | to 25 µL       | to 50 µL       |                      |

| STEP                 | TEMPERATURE (°C) | TIME (s)  |
| -------------------- | ---------------- | --------- |
| Initial Denaturation | 95°C             | 30 sec    |
| (1) Denature         | 95°C             | 15-30 sec |
| (2) Anneal           | 45-68°C (Tm+3)   | 15-60 sec |
| (3) Extend           | 68°C             | 1 min/kb  |
| REPEAT (1-3)         | 35 times         | N/A       |
| Final Extension      | 68°C             | 2 min     |
| Hold                 | 4°C              | Forever   |

[NEB Protocol](https://www.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-thermopol-buffer-m0267)

## Troubleshooting your failed PCR

NEB has a decent [PCR Troubleshooting Guide](https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide).

1. Test different annealing temperatures. If you have no product, try a lower annealing temperature. If you have too many undesirable products, try increasing the annealing temperature by a few degrees.
2. Check your primer design. Double check for primer dimerization, poor primer specificity, and so forth. Re-design primers and try another primer pair.
3. Is the quality of your template good? If your template is a miniprep, check to see if there is genomic contamination using the [NanoDrop](https://github.com/reisalex/salis-lab-protocol-book/tree/453898c9360786eef221e6fffd8409c03a547e50/13_Lab_Equipment/nanodrop.md).
4. If GC-rich templates, try using the polymerase's appropriate GC enhancer.

## A-Tailing with Taq polymerase

*Taq* DNA polymerase, in addition to polymerizing the reverse complement of a given template strand, also adds an additional adenosine residue to the 3'-end of the newly-synthesized strand. This is useful for [T/A cloning](/protocol-book/build-genetic-systems/dna/pcr.md). First, amplify your target DNA with a [high-fidelty DNA polymerase](/protocol-book/build-genetic-systems/dna/pcr.md#-Q5-DNA-polymerase), then A-tail with *Taq*, which is lower-fidelty. After performcing a PCR cleanup on the amplicon, combine the following:

| COMPONENT                 | Volume (µL) |
| ------------------------- | ----------- |
| DNA (PCR cleanup product) | x µL        |
| ThermoPol Buffer          | 5 µL        |
| \*dATP (10 mM)            | 1 µL        |
| *Taq* DNA Polymerase      | 0.2 µL      |
| ddH2O                     | to 50 µL    |

* You can also use 1 µL dNTPs (10 mM), if dATP is not available.

Incubate at 72°C for 20 minutes.

[NEB Protocol](https://www.neb.com/protocols/2013/11/01/a-tailing-with-taq-polymerase)

## Rescue PCR

Rescue PCR is simply a PCR reaction to recover and increase the concentration of a DNA product that you have in very low concentrations. For example, you might do a rescue PCR following a [PCR Assembly](/protocol-book/build-genetic-systems/dna/pcr-assembly.md)