Nucleic Acid Quantification Prt.2 Flashcards
Spectrophotometric Methods
of DNA Quantification
• Nanodrop Method
• Diphenylamine Method
Spectrophotometric Methods of DNA Quantification:
• UV spectrophotometer
• Small volumes (1-2uL)
• No other reagents required
• Quick and easy
Nanodrop Method
Nanodrop Method
• Principle:
• If a 260 nm light source shines on a sample, the amount of
light absorbed by the sample is proportional to the nucleic
acids present in it.
Nanodeop method
• Constant for dsDNA:
• A260 of 1 = 50 ug/mL
Nanodrop
• Calculation of DNA Concentration:
• C = A260 x 50 ug/mL x dilution factor*
Nanodrop method
Advantages
• Can quantify nucleic acid from microvolumes
• Measures DNA, RNA (A260), and protein (A280) concentrations and sample purity (260/280 ratio)
• Large concentration range (2ng/ul – 15,000ng/ul dsDNA) without dilutions
Nanodrop method linearly
2ng/ul – 15,000ng/ul dsDNA
Nanodrop Method
Spectrophotometer Tips
1. Always clean the____ before use
2. Always measure your____
3. Check for___
4.____ your samples before measuring
5. Be wary of ____samples
6. Re-blank the machine every____
7. Take measurements in (at least) duplicates
8. Always write down your results
9. Always clean the pedestal after use
pedestal
blank
air bubbles
Mix
low concentration
30 mins
DNA PURITY
• Ratio of A260/A280 (_____)
• Ratio of A260/A230 (_____)
~1.8
~2.0
DNA concentration
•____ dependent
• add ____ to maintain____
pH
10 mM Tris·Cl
pH 7.5
Constant
A260 of 1.0 =_____ for RNA
40 µg/mL
A260/A280 ratio (~1.8 for pure DNA)
• Measures______ contamination (proteins absorb at 280 nm).
• A low ratio (<1.8) suggests____
• A high ratio (>2.0) suggests possible____
protein
protein contamination.
RNA contamination.
= A230
Inorganic and Organic substances
= A270
Phenol
= A280
Proteins
Optimal A260/A280 Ratio
1.80-2.00
Optimal A260/A230 Ratio
2.00-2.20
is a colorimetric assay used to quantify DNA based on a chemical reaction between deoxyribose and diphenylamine under acidic conditions.
Diphenylamine (DPA) method
Principle of the Diphenylamine Method
• Deoxyribose in DNA undergoes acid hydrolysis, breaking it down into_______.
• This aldehyde reacts with diphenylamine, forming a_____.
• The intensity of this color is directly proportional to the amount of DNA present.
• The absorbance of the complex is measured at_____ using a spectrophotometer.
• DNA concentration is determined by comparing the absorbance to a standard curve.
w-hydroxylevulinic aldehyde
blue-colored complex
595 nm
Method
Sensitivity
Advantages
Limitations
2 ng/uL
Simple, Fast
Low specificity, Contaminants affect accuracy
UV Spectrophotometry (Nanodrop Method)
Method
Sensitivity
Advantages
Limitations
3 µg
Can be used with a colorimeter or ELISA reader
Labor-intensive, Time-consuming
Diphenylamine Method
Method
Sensitivity
Advantages
Limitations
10-50 pg/µL (depends on kit used)
High specificity for nucleic acid type
Expensive reagents required
Fluorescence Measurement
Method
Sensitivity
Advantages
Limitations
10 ng (depends on ladder and imaging)
Very high specificity for DNA size
Time-consuming, Less accurate for concentration
Gel Electrophoresis
