Nucleic acid quantification is essential for assessing the concentration and purity of DNA/RNA, which is critical for applications like PCR and restriction digestion. Two primary methods for quantification are the spectrophotometric method using NanoDrop and agarose gel electrophoresis, each having distinct advantages for measuring sample purity and concentration. The document outlines how to interpret spectrophotometric readings and the separation of nucleic acids in agarose gel, including the identification of contaminants.

1. Nucleic Acid Quantification
DNA / RNA Quantification
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2. Why to quantify Nucleic acids?
• Nucleic acids are quantified to check the concentration and
purity of DNA/RNA present in the solution mixture.
• It is important to know the concentration and purity of the
nucleic acid for the use in further applications like PCR,
restriction digestion etc.
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3. Nucleic acid Quantification
Methods
• Spectrophotometric Mehtod
• Agarose gel electrophoresis
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4. Spectrophotometric Mehtod
NanoDrop 2000c Spectrophotometer
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5. Advantages of using Nano Drop
• Can quantify nucleic acid from micro volumes of 0.5µL –
2.0µL.
• Measures DNA, RNA (A260) and Protein (A280)
concentrations and sample purity (260/280 ratio).
• Large concentration range (2 ng/µL – 15,000 ng/µL dsDNA)
without dilutions.
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6. Purity and Quantification – Nano
Drop
• Pure DNA sample gives a 260/280 ratio ~ 1.8.
• For pure RNA 260/280 ratio is ~ 2.
• A 260/230 Indicates possible contaminats.
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7. Spectra of 4 commonly used
extraction reagents
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8. Checking contaminants in Extracted
sample
• A low 260/230 ratio indicates the contaminants absorbing at
230nm or less.
• A low 260/280 ratio indicates the contaminants absorbing at
280nm or less.
• A shift in the wavelength trough is indicative of contaminants
absorbing at low wavelengths.
• The wavelength of the sample peak should be at 260nm if
contaminants are present the peak may shift.
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9. Comparison of Pure DNA vs
Contaminated DNA Preparation
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10. Agarose Gel Electrophoresis
• It is a separation method used to separate nucleic acids based
on their size under the influence of electric current.
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11. Quantification & Purification of
Nucleic acid
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12. Quantification and Purification
Nucleic Acids
• Contaminating DNA/RNA fragments can be removed using
this method.
• After running the sample on gel the band of interest can be
spliced out and gel extraction can be done to purify it.
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13. Thank You
Bio-Resource
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12/05/nucleic-acid-quantification-
dnarna.html
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