MoBio Lab Review

MoBio Lab Review - Spectrophotometric Analysis of Nucleic...

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    Spectrophotometric Analysis of Nucleic Acids Topics: How to quantify nucleic acids concentration How to assess purity of nucleic acids How to calculate and perform dilutions How to operate spectrophotometer
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    Ultraviolet (UV, 200-400 nm) spectrophotometry can be used to  - determine the quantity of nucleic acids (quantitative)  - estimate nucleic acid purity (qualitative) - amount of energy absorbance is proportional to [substance] Absorption is primarily due to the aromatic rings.            Purine             Pyrimidine   Spectrophotometric Analysis of Nucleic Acids
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    Differences in Nucleotides of DNA and RNA In DNA, the 2’ hydroxyl group of ribose is missing. Uracil is a Thymine without the methyl group
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    Temperature (°C) Absorbance at 260 nm ds DNA ss DNA (weaker interaction between base pairs higher abs) DNA Melting Curve Aromatic bases are stacked in the hydrophobic core
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    A 260 (1.0) = 50 μ g/ml – ds DNA 40 μ g/ml – ss DNA or RNA 33 μ g/ml – oligonucleotides Quantitative Measurement of Nucleic Acid
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    A 260 / A 280 ratio indication of protein contamination (1.8~1.9 for DNA and 1.9~2.0 for RNA) Lower ratios indicate contamination (higher A280 value) A 230 Phenol or Urea contamination A 325 Dirty cuvetes A 260 /A 230 ratio: a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are Qualitative Measurement of Nucleic Acid
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          1 :5 0   1 :1 00   1 :2 00           1 :8 0   1 :2 50   1 :4 00   Sam ple 1   ( μ l)          Sam ple 2   ( μ l)        H 2 ( μ l)          H 2 ( μ l)        Final  Volum e  (m l)  1.0         (m l)          Sample 1 (50 μ l ds DNA) & Sample 2 (50 μ l ss RNA) Make individual dilutions from stock (final volume: 1 ml) How to make a dilution using the dilution factor method : To make a 1:10 dilution of a stock, you will mix 1 part of the stock solution (regardless of its concentration) with 9 parts of diluent to a total of 10 parts. Show us your calculations before making the dilutions!!
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          1 :5 0   1 :1 0 0   1 :2 0 0           1 :8 0   1 :2 5 0   1 :4 0 0   Sam ple 1   ( μ l)          Sam ple 2   ( μ l)        H 2 ( μ l)          H 2 ( μ l)        Final  Volum e  (m l)  1.0         (m l)          Sample 1 (50 μ l ds DNA) & Sample 2 (50 μ l ss RNA) Make individual dilutions (final volume: 1 ml) A 260 A 280 A 230 A 325 Record absorbance at various wavelengths (Blank @ every wavelength with nano water) Record data in table 2 2 groups share 1 instrument. Do not copy each other’s work . Save all samples until  the very end!
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    Expected Concentration: _________ Spectrophotometric Analysis of Nucleic Acids compare compare Sample Dilution A 260 A 280 A 230 A 325 A 260 :A 280 A 260 :A 230 Final  Concentration  (mg/ml) 1 1:50 1 1:100 1 1:200 2 1:80 2 1:250 2 1:400
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    Liquid Waste: Small plastic waste container
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MoBio Lab Review - Spectrophotometric Analysis of Nucleic...

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