PCR TroubleshootingPCR Troubleshooting

DefinitionDefinition

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

ExamplesExamples

PCR troubleshooting is used to…

Increase primer specificityIncrease quantity of PCR productIncrease quality of PCR product

Non-examplesNon-examples

PCR Troubleshooting is not used to…

Purify extracted DNA Create recombinant DNAQuantify DNA

Factors Influencing PCR SuccessFactors 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 StrategiesTroubleshooting 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 StrategiesTroubleshooting Strategies

Extend annealing time

Allows primers more time to find correct complement on template DNA

Can increase quantity of PCR productBut decreased primer specificity can lead

to smeared bands

Troubleshooting StrategiesTroubleshooting 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 StrategiesTroubleshooting Strategies

Other adjustable parameters:

Magnesium Chloride (MgCl2) concentration

DNA template concentrationdNTP concentrationPrimer concentration

END

California State Chemistry StandardsCalifornia 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 StandardsCalifornia 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 California State Investigation and ExperimentationInvestigation and Experimentation

StandardsStandardsGrade 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 StandardsNational Standards

Grades 6-12

Content Standard A: Science as Inquiry

 

Content Standard C: Life Science

Content Standard E: Science and Technology