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Analytical Chem JoVE (Open Access) Citable · DOI

Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies

DOI: 10.3791/3998-v
What you'll learn
  • ✓ Set up and execute a basic PCR reaction from reagent assembly through thermal cycling
  • ✓ Identify and troubleshoot common PCR failures by understanding reaction variables
  • ✓ Optimize PCR conditions to generate desired amplification products
  • ✓ Program thermal cycler parameters for standard PCR protocols
Protocol

PCR has emerged as a common technique in many molecular biology laboratories. Provided here is a quick guide to several conventional PCR protocols. Because each reaction is a unique experiment, optimal conditions required to generate a product vary. Understanding the variables in a reaction will greatly enhance troubleshooting efficiency, thereby increasing the chance to obtain the desired result.

Difficulty
beginner
Total time
~2–3 hours per reaction (including thermal cycling time)
Biosafety
BSL-1

Steps

1
Assemble PCR reagents and materials

Gather all necessary reagents (polymerase, dNTPs, buffer, template DNA, primers) and materials (tubes, pipettes, thermal cycler) before beginning reaction setup.

▶ 01:20
2
Set up polymerase chain reaction mixture

Prepare PCR reaction by combining polymerase, dNTPs, buffer, template DNA, and primers in appropriate proportions and volumes.

▶ 02:24
3
Execute basic PCR protocol

Load prepared reaction mixture into thermal cycler and initiate standard amplification cycles following established PCR protocol.

▶ 04:24
4
Program thermal cycler parameters

Set thermal cycler temperature profiles, cycle numbers, and timing for denaturation, annealing, and extension steps.

▶ 05:12
5
Troubleshoot PCR amplification failures

Diagnose and resolve common PCR problems by systematically adjusting reaction variables such as temperature, reagent concentration, and cycle parameters.

▶ 06:29
6
Amplify target DNA sequence

Apply optimized PCR protocol to amplify Gal3 target gene as a worked example demonstrating successful product generation.

▶ 07:27
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