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DNA Purification

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  1. DNA Purification

  2. Before (b) and after (a) DNA purification DNA Impurities Definition • DNA purification is a technique that removes impurities and unused reagents from samples after enzymatic reactions, such as PCR www1.qiagen.com

  3. Quick Quiz The purpose(s) of DNA purification is to: • Remove excess reagents remaining from PCR • Remove sample impurities • Prepare the sample for downstream applications (i.e. sequencing) • All of the above

  4. Non-examples • DNA purification is not used to… • Isolate DNA from intact cells • Change the concentration of DNA in a given volume • Purify protein or RNA samples

  5. Downstream Applications • Purified PCR products may be used in the following types of reactions… • Additional PCRs • Sequencing • Restriction digestion • Ligation and transformation (Cloning) • Labeling or hybridization

  6. Qiagen PCR Purification Mix sample and buffer. Makes DNA in solution ready to bind column. Load onto column. Nucleic acids bind to membrane. Other components flow through. Wash Column. Removes any contaminants. Elute DNA. DNA releases from column and is ready for downstream applications.

  7. Quick Quiz In column purification, DNA is bound to the column membrane using a ______ salt solution. DNA is eluted off the membrane using a ______ salt solution. • High, low • Low, high • Iodized, non-iodized • Saturated, supersaturated

  8. Quick Quiz Which list of activities is in the correct order? • DNA extraction, DNA purification, PCR, DNA sequencing • DNA purification, DNA extraction, PCR, DNA sequencing • DNA extraction, PCR, DNA purification, DNA sequencing • DNA extraction, PCR, DNA sequencing, DNA purification

  9. END

  10. Resources • Modified QIAquick protocol: “PCR Purification” handout • Qiagen QIAquick PCR purification protocol

  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 ExperimentationStandards Grades 7, 9-12 a/b. Select and use appropriate tools and technology to perform tests, collect data, analyze relationships, and display data Grade 8 a. Plan and conduct a scientific investigation to test a hypothesis

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