1. Why Serial Dilutions Are Preferred To One Serial Dilution
1) Why serial dilutions are preferred to one large dilution in lab experiments?
Answer: Serial dilution is the technique of performing repeated dilutions on the same chemical in order to change its concentration. The diluted solution from a serial dilution can be used to calculate the concentration of the actual solution. In experimental work, often we need to obtain a range of concentrations for a specific compound. Thus, instead of preparing one large dilution, if we take a concentrated sample of a particular compound and perform a series of dilutions with it, we can obtain various concentrations of the same compound. In the context of this experiment, for gel electrophoresis and absorbance spectrophotometry, we need to prepare a series of …show more content…
Therefore, instead of obtaining the absorbance of dsDNA at 260 nm, we obtain the ratio of absorbance of dsDNA at 260 nm and the absorbance of protein at 280 nm (A260/A280 ). If this ratio is 1.8, then we can be sure that dsDNA is pure. But if the reading is less than 1.8, then the dsDNA was contaminated with protein or phenol and if the reading is more than 1.8, then we can be sure that the dsDNA was contaminated with RNA or degraded DNA. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA and the presence of contaminant organic compounds. The lower the ratio, the greater the amount of thiocyanate salt or organic compounds is present, for example. As a guideline, the A260/A230 is best if greater than …show more content…
Fluorescence methods are more sensitive than absorbance, particularly for low-concentration samples, and the use of DNA-binding dyes allows more specific measurement of DNA than spectrophotometric methods allows. Hoechst bisbenzimidazole dyes, PicoGreen® and QuantiFluor™ dsDNA dyes selectively bind double-stranded DNA. The availability of single-tube and microplate fluorometers gives flexibility for reading samples in PCR tubes, cuvettes or multiwell plates and makes fluorescence measurement a convenient modern alternative to the more traditional absorbance methods. As with absorbance methods, dilution factor must be taken into account when calculating DNA concentration from fluorescence values. Materials required for fluorescence methods are: a fluorescent DNA binding dye, a fluorometer to detect the dyes, and appropriate DNA standards. Depending on the dye selected, size qualifications may apply, and the limit of detection may vary. The usual caveats for handling fluorescent compounds also apply—photobleaching and quenching will affect the
Answer: Serial dilution is the technique of performing repeated dilutions on the same chemical in order to change its concentration. The diluted solution from a serial dilution can be used to calculate the concentration of the actual solution. In experimental work, often we need to obtain a range of concentrations for a specific compound. Thus, instead of preparing one large dilution, if we take a concentrated sample of a particular compound and perform a series of dilutions with it, we can obtain various concentrations of the same compound. In the context of this experiment, for gel electrophoresis and absorbance spectrophotometry, we need to prepare a series of …show more content…
Therefore, instead of obtaining the absorbance of dsDNA at 260 nm, we obtain the ratio of absorbance of dsDNA at 260 nm and the absorbance of protein at 280 nm (A260/A280 ). If this ratio is 1.8, then we can be sure that dsDNA is pure. But if the reading is less than 1.8, then the dsDNA was contaminated with protein or phenol and if the reading is more than 1.8, then we can be sure that the dsDNA was contaminated with RNA or degraded DNA. Good-quality DNA will have an A260/A280 ratio of 1.7–2.0. Strong absorbance around 230nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260nm to 230nm can help evaluate the level of salt carryover in the purified DNA and the presence of contaminant organic compounds. The lower the ratio, the greater the amount of thiocyanate salt or organic compounds is present, for example. As a guideline, the A260/A230 is best if greater than …show more content…
Fluorescence methods are more sensitive than absorbance, particularly for low-concentration samples, and the use of DNA-binding dyes allows more specific measurement of DNA than spectrophotometric methods allows. Hoechst bisbenzimidazole dyes, PicoGreen® and QuantiFluor™ dsDNA dyes selectively bind double-stranded DNA. The availability of single-tube and microplate fluorometers gives flexibility for reading samples in PCR tubes, cuvettes or multiwell plates and makes fluorescence measurement a convenient modern alternative to the more traditional absorbance methods. As with absorbance methods, dilution factor must be taken into account when calculating DNA concentration from fluorescence values. Materials required for fluorescence methods are: a fluorescent DNA binding dye, a fluorometer to detect the dyes, and appropriate DNA standards. Depending on the dye selected, size qualifications may apply, and the limit of detection may vary. The usual caveats for handling fluorescent compounds also apply—photobleaching and quenching will affect the