 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

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

 PCR Troubleshooting is not used to…
 Purify extracted DNA
 Create recombinant DNA
 Quantify DNA

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

 Vary temperatures to optimize primer annealing
Cycle Temperatures
94 o 55 o - 72 o
94 o 48 o - 68 o
94 o 45 o - 65 o
94 o 40 o - 65 o
Reasoning
Well-matched primers
Poorly matched primers
“Fishing expedition”
Do these primers work?

 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

 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

 Other adjustable parameters:
 Magnesium Chloride (MgCl
2
) concentration
 DNA template concentration
 dNTP concentration
 Primer concentration

END

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