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? 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

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

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

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

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

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

Hind III digested lambda phage DNA 238ng 97ng 68ng 45ng 24ng 21ng 6ng 1ng 477ng 135ng 194ng 90ng 48ng 42ng 12ng 3ng 1.0ug0.5ug Approx. ng

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

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

Using the ND Turn on PC 2.Open NanoDrop program 3.Select “DNA” from menu

Using the ND Lift arm 2.Place 1 μL water on pedestal to initialize instrument 3.BLANK instrument using water or elution buffer 4.Place 1-2 μL of DNA sample on pedestal 5.Click “MEASURE” on instrument screen 6.Recover sample if precious, or clean pedestals using a lab wipe

NanoDrop Data Screen

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

Quick Quiz The Nanodrop reads: A.1ul of sample at visible wavelengths B.10ul of sample at visible wavelengths C.1 ul of sample at UV wavelengths D.10 ul of sample at UV wavelengths

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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 Thermo Scientific NanoDrop ND1000 User Manual  manual.pdf manual.pdf NanoDrop: It’s Like Hot video 

California State Chemistry Standards Grade 8 6c. Living organisms have many different kinds of molecules… Grades 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 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 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

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