Four Tips for Optimizing Your PCR Amplification

R Amplification, PCR Troubleshooting PCR success can vary greatly depending on your experimental design. Small errors can very often result in low yield or non-specific products. Here are 4 essential tips to ensure the success of your next PCR reaction:

Primer Design

Correct primer design is crucial for precise primer-template annealing and avoiding primer-dimer formation. Primer-dimer amplification can lead to competition for PCR reagents and inhibit your reaction.

  • Avoid sequence complexity. To prevent secondary structure formation, a primer length of 18-30 nucleotides and GC content of 40-60% is recommended. Also avoid stretches of 4+ nucleotide and dinucleotide repeats.
  • Check for primer homology. Any annealing between or internally within your primers will result in reduced PCR efficiency.
  • Match primer Tm. Design both primers to have melting temperatures within 3°C of each other to simplify your PCR optimization.
  • End with a G or C. Capping the 3' end of your primer sequence with a G or C will strengthen primer annealing at the site of extension.
  • Remember to add spacers for restriction enzyme cloning/isothermal assembly. While this will not affect the efficiency of your reaction, restriction enzymes require at least 6 terminal base pairs for binding, while isothermal assembly requires a homology region of at least 15 base pairs.
  • Maintain proper primer concentrations. A concentration of 0.3-1.0 µM is recommended, excessive quantities may lead to primer-dimer formation.

Template Sequence

A PCR reaction can be affected by both the quality and quantity of your DNA template.

  • A "clean" template will increase reaction specificity and product yield. Be careful to avoid contamination during template extraction. Protein or chemical contamination can result in non-specific background or outright kill your PCR reaction. Check that your DNA absorbance has a 260nm/280nm ratio of ≥1.8.
  • Less is more. Start small with the amount of your template to reduce the chances for non-specific annealing. We recommend using 1 ng when amplifying plasmid DNA and 100 ng when amplifying genomic DNA.
  • Check the GC-content of your template. DNA templates enriched with over 60% GC-content have increased stability and may require additional reagents for separation. There are a number of GC-rich chemical enhancer options, including 5% DMSO, 1M ethylene glycol, and 0.8M 1,2-propanediol.

Reaction Reagents

While compromised reagents can affect the efficiency of your PCR reaction, most reagents hold up well with lab use. Here are the components to consider:

  • DNA Polymerase- Remember to avoid multiple freeze-thaw cycles to maintain the quality of your polymerase. An amount of 0.2-0.5 µL is recommended for most standard reaction volumes.
  • dNTPs- Remember to avoid multiple freeze-thaw cycles to prevent the degradation of your dNTPs. A final concentration of between 50-200 µM is recommended, as too much can actually inhibit your PCR reaction.
  • Magnesium- A concentration of 1.5-2.0 mM is recommended, but optimal conditions may vary depending on your polymerase.

Thermocycler Conditions

Thermocycler protocols and conditions are fairly standardized for PCR. Here are the three major aspects to consider:

  • Modified PCR Setups- For complex primer and template sequences, consider either a hot start or touchdown PCR setup. Hot-start PCR reduces non-specific priming, by utilizing a modified polymerase, which requires an initial activation at 95°C. Touchdown PCR increases the initial annealing temperature beyond the optimal Tm, this temperature is gradually reduced in later cycles to more permissive levels. This process promotes selective amplification of the desired product.
  • Annealing Temperature- We recommended setting your annealing temperature at 3°C below the melting temperature of your primer sequence melting point. When calculating melting temperature, remember to exclude any primer regions that do not anneal to the template. For future optimization, annealing temperature can be increased in 1-2°C steps.
  • Extension Time- We recommend setting 1 minute of extension time per 1kb of amplicon. Optimal extension rates may depend on the processivity of the DNA polymerase.

Molecular Cloning Workflow

In molecular cloning, ligation of a DNA template into a vector is confirmed through bacterial transformation, screening, and sequencing.

Molecular Cloning Workflow. In molecular cloning or PCR cloning, the DNA template of interest is first amplified and modified by PCR to add necessary restriction enzyme sequences. Digestion of purified DNA template and plasmid with the specified restriction enzyme(s) allows ligation of the DNA template into the open vector. Bacterial transformation supports screening of individual colonies for the desired vector containing the new DNA insert, which may be performed through sequencing following plasmid extraction.

  • Don, R., Cox, P., Wainwright, B., Baker, K. & Mattick, J. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research 19, 4008-4008 (1991).
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