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Quantifying Sample DNA

Quantifying Sample DNA. Definition. Quantifying DNA: a technique to calculate the quantity (weight) of DNA (deoxyribonucleic acid) in a sample. Using a known volume of sample allows you to calculate the concentration (weight per volume). Why Quantify DNA?.

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Quantifying Sample DNA

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  1. Quantifying Sample DNA

  2. Definition • Quantifying DNA: a technique to calculate the quantity (weight) of DNA (deoxyribonucleic acid) in a sample. • Using a known volume of sample allows you to calculate the concentration (weight per volume).

  3. Why Quantify DNA? • For enzymatic reactions, efficiency is dependent on the concentration of all components • This includes the DNA template in PCR • Template DNA is the greatest source of potentially deleterious contaminants (more DNA equals more contaminants) • Too much DNA binds up available Mg++ • Too little DNA means not enough final product

  4. Examples • DNA can be quantified from the following samples • Genomic DNA • PCR product • Plasmid DNA • DNA in a single band after restriction digestion

  5. Non-example • DNA quantification is not used to… • Determine the length (size) of a DNA band

  6. Downstream Applications Quantified DNA may be used in… • PCRs (polymerase chain reactions) • Sequencing reactions • Restriction digestions • Ligations • Bacterial transformations

  7. Quick Quiz Why is it necessary to quantify DNA concentration? • To ensure the proper amount of template gets used in downstream applications • Enzymatic reactions are only successful if all reagents are added in the proper proportions • DNA sequencing requires DNA quantification • All of the above

  8. DNA Quantification: Gels • Molecular weight markers are DNA fragments of known size • Comparison of sample bands to markers allows… • Visible confirmation of desired product • Quantification of sample DNA http://www.biomedcentral.com/content/figures/1471-2180-5-63-2.jpg

  9. 0.5ug 1.0ug 238ng 477ng 97ng 194ng 68ng 135ng 45ng 90ng 21ng 48ng 24ng 42ng 6ng 12ng 1ng 3ng Hind III digested lambda phage DNA Approx. ng Approx. ng 250 500 100 200 50 25 10

  10. Quantifying DNA: Spectrophotometry • Maximum absorption of DNA at 280 nm • Convert absorption to OPTICAL DENSITY (OD) • OD = absorption X dilution factor • 1 OD = 50 mg dsDNA per ml • Generally need large sample size Images from Wikipedia

  11. NanoDrop ND1000 Small sample size: only 1 – 2 μL! No sample dilution: measures up to 3700 ng/ul Fast and easy to use Stores data on computer Converts OD to molarity Quantifying DNA: Spectrophotometry

  12. Turn on PC Open NanoDrop program Select “DNA” from menu Using the ND1000 http://www.nanodrop.com/nd-1000-software.html

  13. Lift arm Place 1 μL water on pedestal to initialize instrument BLANK instrument using water or elution buffer Place 1-2 μL of DNA sample on pedestal Click “MEASURE” on instrument screen Recover sample if precious, or clean pedestals using a lab wipe Using the ND1000 http://www.nanodrop.com/nd-1000-software.html

  14. NanoDrop Data Screen

  15. Spectrophotometer How does it work? Fiber optic cable in measurement arm Sample (1-2 μL) Receiving fiber (fiber optic cable) Xenon lamp

  16. Quick Quiz The Nanodrop reads: • 1ul of sample at visible wavelengths • 10ul of sample at visible wavelengths • 1 ul of sample at UV wavelengths • 10 ul of sample at UV wavelengths

  17. END

  18. Resources • Quantifying DNA Handout • Gel Electrophoresis Handout • DNA Science: A First Course in Recombinant DNA Technology. by David A. Micklos & Greg A. Freyer. Cold Spring Harbor Laboratory Press. 1990. • Thermo Scientific NanoDrop ND1000 User Manual • http://www.nanodrop.com/pdf/NanoDrop%201000-users-manual.pdf • NanoDrop: It’s Like Hot video • http://www.nanodrop.com/nd-1000-nanodrop-it-like-its-hot.html

  19. 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

  20. 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

  21. 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

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

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