PCR Troubleshooting

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PCR Troubleshooting
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Definition

• PCR troubleshooting is a collection of techniques
  that alter PCR reactions in order to achieve
  optimum PCR results

http//academic.brooklyn.cuny.edu/biology/bio4fv/p
age/genetic-engin/pcr.html
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Examples

• PCR troubleshooting is used to
• Increase primer specificity
• Increase quantity of PCR product
• Increase quality of PCR product

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Non-examples

• PCR Troubleshooting is not used to
• Purify extracted DNA
• Create recombinant DNA
• Quantify DNA

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Factors Influencing PCR Success

• Tissue type used for DNA extraction
• Quantity and quality of DNA
• Length of the DNA fragment to be amplified
• Primer specificity

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Troubleshooting Strategies

• Vary temperatures to optimize primer annealing

Cycle Temperatures Reasoning
94o - 55o - 72o Well-matched primers
94o - 48o - 68o Poorly matched primers
94o - 45o - 65o Fishing expedition
94o - 40o - 65o Do these primers work?
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Troubleshooting Strategies

• Extend annealing time
• Allows primers more time to find correct
  complement on template DNA
• Can increase quantity of PCR product
• But decreased primer specificity can lead to
  smeared bands

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Troubleshooting Strategies

• Ramping Gradual increase from annealing to
  extension temperature
• Taq polymerase has more time to extend
  amplification before the optimum extension
  temperature is reached
• Locks primers into position for the final
  extension step

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Troubleshooting Strategies

• Other adjustable parameters
• Magnesium Chloride (MgCl2) concentration
• DNA template concentration
• dNTP concentration
• Primer concentration

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• END

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California State Chemistry Standards

• Grade 8
• 6c. Living organisms have many different kinds
  of molecules
• Grades 9-12
• 6. Solutions are homogenous mixtures of two or
  more substances
• 7. Energy is exchanged or transformed in all
  chemical reactions and physical changes of matter
• 8. Chemical reaction rates depend on factors that
  influence the frequency of collision of reactant
  molecules

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California State Biology Standards

• Grade 7
• 2e. DNA is the genetic material of living
  organisms and is located in the chromosomes of
  each cell
• 3a. Genetic variation and environmental factors
  are causes of evolution and diversity of
  organisms
• Grades 9-12
• 2. Mutation and sexual reproduction lead to
  genetic variation in a population
• 7. The frequency of an allele in a gene pool of
  a population depends on many factors and may be
  stable or unstable over time

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California State Investigation and
ExperimentationStandards

• Grade 7-8
• a. Select and use appropriate tools and
  technology to perform tests, collect data,
  analyze relationships, and display data
• Grades 9-12
• c. Identify possible reasons for inconsistent
  results, such as sources of error or uncontrolled
  conditions
• d. Formulate explanations by using logic and
  evidence
• k. Recognize the cumulative nature of
  scientific evidence
• l. Analyze situations and solve problems that
  require combining and applying concepts from more
  than one area of science

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National Standards

• Grades 6-12
• Content Standard A Science as Inquiry
•  
• Content Standard C Life Science
• Content Standard E Science and Technology