6 Questions
What is the general recommendation to avoid inhibition when re-amplifying PCR products?
Dilute the reaction in water before the next round of PCR
How much gDNA template is recommended per 50-µl reaction volume?
5-50 ng
What is the risk associated with using higher amounts of DNA when amplifying PCR targets?
Increase nonspecific amplification risk
Why is it important to optimize DNA input when performing PCR?
To avoid excessively high DNA concentrations
What type of DNA polymerase would require less input DNA due to higher sensitivity to the template?
Polymerase engineered for higher sensitivity
What are the adverse effects of excessively high starting DNA concentrations during PCR?
Inhibit amplification reactions
Study Notes
PCR Template Requirements
- PCR template can be derived from any DNA source, including genomic DNA (gDNA), complementary DNA (cDNA), and plasmid DNA.
PCR Optimization
- Re-amplification of PCR products can be done to increase target yield, but carryover reaction components can inhibit amplification.
- To avoid inhibition, dilute the reaction in water before the next round of PCR or purify PCR amplicons before re-amplification.
Template Amounts
- Recommended amount of gDNA template is 5-50 ng per 50-µl reaction volume.
- For plasmid PCR targets, use 0.1-1 ng per 50-µl reaction volume.
- Optimal template amounts vary based on the type of DNA polymerase used, with more sensitive polymerases requiring less input DNA.
Importance of Optimization
- Lower template amounts reduce yields.
- Higher template amounts increase the risk of nonspecific amplification.
- Excessively high concentrations of starting DNA (> 500-1000 ng) can inhibit amplification reactions.
Learn about the various DNA sources that can be used for PCR template amplification, such as gDNA, cDNA, and plasmid DNA. Understand the importance of avoiding inhibition by diluting the reaction in water or re-amplifying PCR products for higher yield.
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