 |
| Thermofisher, Nanodrop 1000 |
Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm).
Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument.
It works on the principle of Spectrophotometer or Sample Retention System....
Principle of Nanodrop:
"The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of column and forms approximately 1mm optical path length for the ultra violet light. When UV illuminates the sample, the wavelength emitted by the sample will be recorded by the detectors which will produce the digital data in the form wavelength ratios. These ratios and plots represent the average concentrations of DNA or RNA present in the sample and their purity".
Nucleic Acid Quantification:
"Some of the molecular biology experiments or reactions require nucleic acids with particular amounts and purity for the optimal performance. The nucleic acid quantification is commonly performed for the determination of the concentrations of DNA or RNA present in a mixture and to determine if there are any contaminants present in the sample mixture. For example, the cloning experiments, usually required proper amount of DNA of interest with good purity for better clones. Commonly used quantification method is by using the spectrophotometer analysis. Usually, the quantification is performed by determining the absorbance of UV light at 260nm, 230nm, 280nm wavelengths. (Usually between, 2400nm to 350nm).. Ratios of the absorbance wavelengths are used to calculate the concentrations. (Nucleotides, RNA, dsDNA and ssDNA all absorb at 260nm and they contribute to the total absorbance of the given sample.
For DNA and RNA, the quantification is done by considering the ratios of the absorbance at 260nm and absorbance at 280nm.
Determining the Concentrations (How to calculate absorbance ratio):
Common technique to measure the concentrations of the nucleic acids is by determining the absorbance at 260nm and 280nm and their ratios.
Ratio = (Absorbance at 260nm) / (Absorbance at 280nm).
For DNA, ratio of ~1.9 is accepted for the pure DNA.
and
For RNA, ratio of ~2.0 is accepted for the pure RNA
Principle of Nanodrop:
"The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of column and forms approximately 1mm optical path length for the ultra violet light. When UV illuminates the sample, the wavelength emitted by the sample will be recorded by the detectors which will produce the digital data in the form wavelength ratios. These ratios and plots represent the average concentrations of DNA or RNA present in the sample and their purity".
Nucleic Acid Quantification:
"Some of the molecular biology experiments or reactions require nucleic acids with particular amounts and purity for the optimal performance. The nucleic acid quantification is commonly performed for the determination of the concentrations of DNA or RNA present in a mixture and to determine if there are any contaminants present in the sample mixture. For example, the cloning experiments, usually required proper amount of DNA of interest with good purity for better clones. Commonly used quantification method is by using the spectrophotometer analysis. Usually, the quantification is performed by determining the absorbance of UV light at 260nm, 230nm, 280nm wavelengths. (Usually between, 2400nm to 350nm).. Ratios of the absorbance wavelengths are used to calculate the concentrations. (Nucleotides, RNA, dsDNA and ssDNA all absorb at 260nm and they contribute to the total absorbance of the given sample.
For DNA and RNA, the quantification is done by considering the ratios of the absorbance at 260nm and absorbance at 280nm.
Determining the Concentrations (How to calculate absorbance ratio):
Common technique to measure the concentrations of the nucleic acids is by determining the absorbance at 260nm and 280nm and their ratios.
Ratio = (Absorbance at 260nm) / (Absorbance at 280nm).
For DNA, ratio of ~1.9 is accepted for the pure DNA.
 |
| DNA Plot Example |
and
For RNA, ratio of ~2.0 is accepted for the pure RNA
 |
| RNA Plot Example |
Comments
Post a Comment