1. PCR Troubleshooting

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

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

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

5. 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

6. 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?

7. 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

8. 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

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

10. END

11. 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

12. 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

13. 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

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