BenchMasteryMolecular biology techniques & virtual lab

PCR

Amplify a specific DNA region from a small amount of template.

ampliconSimulator readyFull simulatorEngine: Thermal cycling & amplification → Electrophoresis & blotting
Choose a sub-technique
In learning pathsClone a gene · step 1 of 8 · next: Agarose gel electrophoresis →

Our ruleSimulations prepare you for the bench but don't replace hands-on practice.

In this lesson · about 2 min read
  1. How PCR works
  2. Why it's exponential
  3. What goes in the tube
  4. How the gel works
  5. Key terms
  6. When to use it
  7. Sub-techniques

1How PCR works

The polymerase chain reaction copies one stretch of DNA, defined by two short primers, over and over. Each cycle has three temperature steps.

1. Denature95 °Cstrands separate2. Anneal~56 °Cprimerprimer3. Extend72 °Cpolymerase builds 5′→3′
One cycle: heat separates the strands, primers bind their sites as the block cools, and the polymerase extends each primer into a new strand.
94–98 °C · 15–30 s

1. Denature

Heat breaks the hydrogen bonds between strands, so the double helix separates into two single strands.

50–65 °C · 15–30 s

2. Anneal

Cooling lets the primers bind their complementary sites. Usually set 3–5 °C below the primers' Tm. Too low and primers bind wrong sites; too high and they barely bind.

72 °C · ~1 min per kb

3. Extend

The polymerase adds nucleotides from each primer's 3′ end, building the new strand. Taq works best near 72 °C.

2Why it's exponential

After n perfect cycles one template gives 2ⁿ copies: about a thousand after 10 cycles, a million after 20 and a billion after 30. Real reactions run at roughly 90–95% efficiency and then plateau as primers and dNTPs are used up and the polymerase tires. The virtual lab's chart shows this curve for your own run.

10³10⁶10⁹10¹²visible on gelexponentialplateau0102030cycle
Copies double each cycle until the reaction runs out of steam and plateaus. A band only shows on a gel once there are roughly 10¹⁰ copies (~5 ng).

3What goes in the tube

Template
The DNA containing your target, here human genomic DNA (about 300 genome copies per nanogram).
Primers
Two short oligonucleotides (18–25 nt) flanking the target, one per strand. They define what gets copied.
Polymerase
A heat-stable DNA polymerase such as Taq. High-fidelity enzymes make fewer errors when the sequence matters.
dNTPs
The four nucleotide building blocks (dATP, dCTP, dGTP, dTTP).
Mg²⁺
Polymerase cofactor. Too little gives weak product; too much stabilizes mismatched primers and gives extra bands.
Buffer
Keeps pH and salt right for the enzyme.

4How the gel works

DNA's phosphate backbone is negatively charged, so in an electric field it moves toward the positive electrode. The agarose mesh slows long molecules more than short ones, so fragments separate by size.

Key terms

Practise these as flashcards →
Tm (melting temperature)

The temperature at which half of a primer is bound to its target. Annealing is set relative to it.

Amplicon

The product of a PCR: the stretch of DNA between and including the two primers.

NTC (no-template control)

A tube with every reagent except template. It must stay blank; a band means contamination.

Primer dimer

A short artefact (~40–60 bp) made when primers anneal to each other and get extended.

Plateau

The late phase where product stops doubling because reagents run low and the enzyme loses activity.

Mispriming

Primers binding partly matching sites, which gives off-target bands.

Fidelity

How rarely the polymerase inserts a wrong base. Proofreading enzymes are roughly 50–300× more accurate than Taq.

When to use it

  • You need many copies of a known DNA region: for cloning, sequencing, genotyping or as a probe.
  • You want a yes/no answer for whether a sequence is present (a band or no band).
  • Endpoint PCR is not quantitative: use qPCR or digital PCR when you need to know how much target there is.

Compared with alternatives

Quantitative PCR (qPCR) →

Measures product every cycle with a fluorescent dye or probe, so it quantifies the starting amount (Ct). No gel needed.

Digital PCR →

Splits the reaction into thousands of partitions and counts positives for absolute copy numbers, useful for rare mutations.

Isothermal amplification →

Amplifies at one temperature (LAMP, RPA), so it works without a thermocycler for field or point-of-care tests.

Reverse transcription →

Turns RNA into cDNA first when the target is a transcript (RT-PCR).

Agarose gel electrophoresis →

The standard readout for endpoint PCR: confirms size and specificity of the product.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Uses a proofreading polymerase with a 3′→5′ exonuclease that removes mismatched bases, giving roughly 50× or more fewer errors than Taq. Use it whenever the sequence matters: cloning, mutagenesis, sequencing templates.

  • Denature at 98 °C and keep steps short; these enzymes are fast (15–30 s per kb).
  • Add the enzyme last: without dNTPs the exonuclease chews primers.
  • Products are blunt-ended, with no 3′ A overhang for TA cloning.
  • Excess Mg²⁺ and many cycles both reduce the fraction of error-free molecules.