PCR Troubleshooting

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/page/genetic-engin/pcr.html

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

Non-examples PCR Troubleshooting is not used to… Purify extracted DNA Create recombinant DNA Quantify DNA

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

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?

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

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

Troubleshooting Strategies Other adjustable parameters: Magnesium Chloride (MgCl2) concentration DNA template concentration dNTP concentration Primer concentration

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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

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

California State Investigation and Experimentation Standards 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

National Standards Grades 6-12 Content Standard A: Science as Inquiry   Content Standard C: Life Science Content Standard E: Science and Technology