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Abstract: Spectrophotometer has become a routine instrument in modern molecular biology laboratories. It is commonly used for quantification of nucleic acids, protein quantification, and bacterial growth concentrations.
The simple principle of spectrophotometer Spectrophotometer uses a light source that can generate multiple wavelengths, through a series of spectrometers to generate a specific wavelength light source. After the light source passes through the tested sample, part of the light source is absorbed to calculate the absorbance value of the sample. This translates into the concentration of the sample. The absorbance of the sample is proportional to the sample concentration.
Quantification of Nucleic Acids The quantification of nucleic acids is the most frequently used function of a spectrophotometer. Oligonucleotides, single-stranded, double-stranded DNA, and RNA can be quantified in buffers. The absorption peak of the highest absorption peak of nucleic acid is 260 nm. Each nucleic acid has a different molecular composition and therefore has different conversion factors. To quantify different types of nucleic acids, select the corresponding coefficients in advance. For example, the absorbance of 1OD corresponds to 50 μg/ml dsDNA, 37 μg/ml ssDNA, 40 μg/ml RNA, and 30 μg/ml Olig, respectively. The absorbance value after the test is converted by the above coefficient to obtain the corresponding sample concentration. Prior to testing, select the correct procedure, enter the volume of the stock solution and diluent, and then test the blank and sample solutions. However, the experiment was not easy. Unstable readings may be the biggest headache for experimenters. The higher the sensitivity of the instrument, the greater the exhibited absorbance drift.
In fact, the design principle and working principle of the spectrophotometer allow the absorbance value to change within a certain range, that is, the instrument has a certain degree of accuracy and accuracy. The accuracy of the Eppendorf Biophotometer is ≤ 1.0% (1A). The results of these multiple tests vary between the mean of 1.0% and are all normal. In addition, the physicochemical properties of the nucleic acid, the pH of the buffer for dissolving the nucleic acid, the ion concentration, etc. must also be taken into consideration. During the test, the ion concentration is too high, which can also lead to drifting of the reading. Therefore, it is recommended to use a certain pH value and a low ion concentration. Buffers, such as TE, can greatly stabilize readings. The diluted concentration of the sample is also a non-negligible factor: due to the inevitable presence of small particles in the sample, especially nucleic acid samples. The presence of these small particles interferes with the test effect. In order to minimize the influence of the particles on the test results, it is required that the absorbance of the nucleic acid is at least greater than 0.1 A, and the absorbance is preferably 0.1-1.5 A. In this range, the interference of the particles is relatively small and the result is stable.
This means that the sample concentration should not be too low or too high (exceeding the photometer's test range). The last is the operating factors, such as sufficient mixing, otherwise the absorbance value is too low, or even negative; the mixture can not exist bubbles, the blank solution has no suspended matter, otherwise the reading drifts violently; the same colorimetric cup must be used to test the blank solution and sample , otherwise the concentration difference is too large; the conversion factor and the sample concentration unit are the same; the cuvette that wears the window cannot be used; the volume of the sample must reach the minimum volume required by the color cup, etc.
In addition to nucleic acid concentration, spectrophotometers simultaneously display several very important ratios to indicate the purity of the sample, such as the A260/A280 ratio, for the purpose of assessing the purity of the sample, since the absorption peak of the protein is 280 nm. Pure samples have a ratio greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is lower than 1.8 or 2.0, it indicates the presence of protein or phenolic substances. A230 indicates that there are some contaminants in the sample, such as carbohydrates, peptides, phenols, etc. The ratio of the pure nucleic acid A260/A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. Pure sample, A320 is generally 0.
Direct quantification of proteins (UV method)
This method is to test the protein directly at a wavelength of 280 nm. Select Warburg formula, photometer can directly show the concentration of the sample, or select the appropriate conversion method, the absorbance value is converted to sample concentration.
The protein determination process is very simple. Test the blank first and then test the protein directly. Due to the presence of some impurities in the buffer, the "background" information at 320 nm is generally eliminated and the function "on" is set. Similar to the test nucleic acid, the absorbance value of A280 is required to be at least greater than 0.1 A, and the optimal linear range is between 1.0-1.5. When the Warburg formula was used to show the sample concentration in the experiment, the reading was found to "drift." This is a normal phenomenon. In fact, as long as the observation of the absorbance value of A280 does not exceed 1%, the result is very stable.
The drift is due to the fact that the absorbance value of the Warburg formula is converted to concentration and multiplied by a certain factor. As long as there is a slight change in the absorbance value, the concentration will be amplified and the result will be very unstable.
Protein direct quantification methods are suitable for testing relatively pure, relatively single-component proteins. UV direct quantification is faster and easier to perform than colorimetry; however, it is susceptible to interference from parallel substances, such as DNA interference, and low sensitivity requires high protein concentrations.
Colorimetric protein quantitation Proteins are usually compounds of a variety of proteins. Colorimetric assays are based on protein constituents: amino acids (eg, tyrosine, serine) react with an additional chromogenic group or dye to produce a colored material. The concentration of the colored material is directly related to the number of amino acids the protein reacts, thereby reflecting the protein concentration.
Colorimetry methods are generally BCA, Bradford, Lowry and other methods.
Lowry method: Based on the earliest Biuret reaction and improved. The protein reacts with Cu2 to produce a blue reaction. However, the Lowry method is more sensitive than Biuret. The disadvantage is that several different reagents need to be added sequentially; the reaction takes a long time; it is susceptible to non-protein substances; proteins containing EDTA, Tritonx-100, and ammonia sulfate are not suitable for this method.
BCA (Bicinchoninine acidassay) method: This is a newer, more sensitive protein test method. The protein to be analyzed reacts with Cu2 in an alkaline solution to produce Cu, which forms a chelate with BCA to form a purple compound with an absorption peak at 562 nm. The linear relationship between the concentration of this compound and the protein is extremely strong, and the compound formed after the reaction is very stable. Relative to the Lowry method, the operation is simple and the sensitivity is high. However, similar to the Lowry method, it is susceptible to interference with proteins and detergents.
The Bradford method: The principle of this method is that the protein reacts with Coomassie Brilliant Blue and the resulting colored compound absorbs at 595 nm. Its greatest feature is its high sensitivity, which is twice that of the Lowry and BCA test methods; it is simpler and faster; it requires only one reagent; the compound can be stable for one hour, facilitating results; and with a series of Interfering with Lowry, the BCA reaction reducing agent (eg DTT, mercaptoethanol) is compatible. However, it is still sensitive to detergents. The main drawback is that different standards can lead to large differences in the results of the same sample and no comparable results.
Some researchers who are exposed to a colorimetric method for the first time may be confused by the results of the various colorimetric methods. Which method do you believe to believe? Because of the variety of reactions and color groups Therefore, using several methods at the same time is not comparable to the sample concentration obtained from the same sample. For example, Keller et al. tested the protein in human milk and found that the concentrations measured by Lowry and BCA were significantly higher than that of Bradford. The difference was significant. Even if the same sample is determined, the standard sample selected by the same colorimetric method is inconsistent, and the concentration after the test is also inconsistent. For example, use Lowry to test the protein in the cell homogenate, and use BSA as a standard product with a concentration of 1.34 mg/ml and a globulin as a standard with a concentration of 2.64 mg/ml. Therefore, before selecting a colorimetric method, it is preferable to refer to the chemical composition of the sample to be tested, and find a chemically similar standard protein as a standard. In addition, the colorimetric method for quantifying proteins often causes problems in that the absorbance value of the sample is too low, resulting in a large difference between the measured sample concentration and the actual concentration. The key problem is that the color after the reaction has a certain half-life, so each colorimetric method lists the reaction test time. All samples (including standard samples) must be tested within this time. If the time is too long, the absorbance value obtained becomes smaller and the converted concentration value decreases. In addition, the reaction temperature, the pH value of the solution, etc. are all important factors that affect the experiment. In addition, it is very important to use plastic colorimetry. Avoid using cuvettes made of quartz or glass because the color after reaction will cause quartz or glass to be colored, resulting in inaccurate absorbance of the sample.
Bacterial cell density (OD600)
The laboratory determines the growth density and growth period of the bacteria, and infers the growth density of bacteria based on experience and visual inspection. In the face of demanding experiments, the use of a spectrophotometer to accurately determine the bacterial cell density. OD600 is the standard method for tracking microbial growth in liquid cultures. As a blank solution, an uncultured culture solution was used, and the culture solution containing the bacteria was quantitatively cultured. To ensure correct operation, cell counts must be performed with a microscope for each microorganism and each instrument to make a calibration curve. Occasionally, the negative OD value of the broth was observed in the experiment. The reason was that a color-developing medium was used. After the bacterium was cultured for a period of time, it reacted with the medium to cause a discoloration reaction. In addition, it should be noted that the test sample cannot be centrifuged to keep the bacteria suspended.
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The simple principle of spectrophotometer Spectrophotometer uses a light source that can generate multiple wavelengths, through a series of spectrometers to generate a specific wavelength light source. After the light source passes through the tested sample, part of the light source is absorbed to calculate the absorbance value of the sample. This translates into the concentration of the sample. The absorbance of the sample is proportional to the sample concentration.
Quantification of Nucleic Acids The quantification of nucleic acids is the most frequently used function of a spectrophotometer. Oligonucleotides, single-stranded, double-stranded DNA, and RNA can be quantified in buffers. The absorption peak of the highest absorption peak of nucleic acid is 260 nm. Each nucleic acid has a different molecular composition and therefore has different conversion factors. To quantify different types of nucleic acids, select the corresponding coefficients in advance. For example, the absorbance of 1OD corresponds to 50 μg/ml dsDNA, 37 μg/ml ssDNA, 40 μg/ml RNA, and 30 μg/ml Olig, respectively. The absorbance value after the test is converted by the above coefficient to obtain the corresponding sample concentration. Prior to testing, select the correct procedure, enter the volume of the stock solution and diluent, and then test the blank and sample solutions. However, the experiment was not easy. Unstable readings may be the biggest headache for experimenters. The higher the sensitivity of the instrument, the greater the exhibited absorbance drift.
In fact, the design principle and working principle of the spectrophotometer allow the absorbance value to change within a certain range, that is, the instrument has a certain degree of accuracy and accuracy. The accuracy of the Eppendorf Biophotometer is ≤ 1.0% (1A). The results of these multiple tests vary between the mean of 1.0% and are all normal. In addition, the physicochemical properties of the nucleic acid, the pH of the buffer for dissolving the nucleic acid, the ion concentration, etc. must also be taken into consideration. During the test, the ion concentration is too high, which can also lead to drifting of the reading. Therefore, it is recommended to use a certain pH value and a low ion concentration. Buffers, such as TE, can greatly stabilize readings. The diluted concentration of the sample is also a non-negligible factor: due to the inevitable presence of small particles in the sample, especially nucleic acid samples. The presence of these small particles interferes with the test effect. In order to minimize the influence of the particles on the test results, it is required that the absorbance of the nucleic acid is at least greater than 0.1 A, and the absorbance is preferably 0.1-1.5 A. In this range, the interference of the particles is relatively small and the result is stable.
This means that the sample concentration should not be too low or too high (exceeding the photometer's test range). The last is the operating factors, such as sufficient mixing, otherwise the absorbance value is too low, or even negative; the mixture can not exist bubbles, the blank solution has no suspended matter, otherwise the reading drifts violently; the same colorimetric cup must be used to test the blank solution and sample , otherwise the concentration difference is too large; the conversion factor and the sample concentration unit are the same; the cuvette that wears the window cannot be used; the volume of the sample must reach the minimum volume required by the color cup, etc.
In addition to nucleic acid concentration, spectrophotometers simultaneously display several very important ratios to indicate the purity of the sample, such as the A260/A280 ratio, for the purpose of assessing the purity of the sample, since the absorption peak of the protein is 280 nm. Pure samples have a ratio greater than 1.8 (DNA) or 2.0 (RNA). If the ratio is lower than 1.8 or 2.0, it indicates the presence of protein or phenolic substances. A230 indicates that there are some contaminants in the sample, such as carbohydrates, peptides, phenols, etc. The ratio of the pure nucleic acid A260/A230 is greater than 2.0. A320 detects the turbidity of the solution and other interference factors. Pure sample, A320 is generally 0.
Direct quantification of proteins (UV method)
This method is to test the protein directly at a wavelength of 280 nm. Select Warburg formula, photometer can directly show the concentration of the sample, or select the appropriate conversion method, the absorbance value is converted to sample concentration.
The protein determination process is very simple. Test the blank first and then test the protein directly. Due to the presence of some impurities in the buffer, the "background" information at 320 nm is generally eliminated and the function "on" is set. Similar to the test nucleic acid, the absorbance value of A280 is required to be at least greater than 0.1 A, and the optimal linear range is between 1.0-1.5. When the Warburg formula was used to show the sample concentration in the experiment, the reading was found to "drift." This is a normal phenomenon. In fact, as long as the observation of the absorbance value of A280 does not exceed 1%, the result is very stable.
The drift is due to the fact that the absorbance value of the Warburg formula is converted to concentration and multiplied by a certain factor. As long as there is a slight change in the absorbance value, the concentration will be amplified and the result will be very unstable.
Protein direct quantification methods are suitable for testing relatively pure, relatively single-component proteins. UV direct quantification is faster and easier to perform than colorimetry; however, it is susceptible to interference from parallel substances, such as DNA interference, and low sensitivity requires high protein concentrations.
Colorimetric protein quantitation Proteins are usually compounds of a variety of proteins. Colorimetric assays are based on protein constituents: amino acids (eg, tyrosine, serine) react with an additional chromogenic group or dye to produce a colored material. The concentration of the colored material is directly related to the number of amino acids the protein reacts, thereby reflecting the protein concentration.
Colorimetry methods are generally BCA, Bradford, Lowry and other methods.
Lowry method: Based on the earliest Biuret reaction and improved. The protein reacts with Cu2 to produce a blue reaction. However, the Lowry method is more sensitive than Biuret. The disadvantage is that several different reagents need to be added sequentially; the reaction takes a long time; it is susceptible to non-protein substances; proteins containing EDTA, Tritonx-100, and ammonia sulfate are not suitable for this method.
BCA (Bicinchoninine acidassay) method: This is a newer, more sensitive protein test method. The protein to be analyzed reacts with Cu2 in an alkaline solution to produce Cu, which forms a chelate with BCA to form a purple compound with an absorption peak at 562 nm. The linear relationship between the concentration of this compound and the protein is extremely strong, and the compound formed after the reaction is very stable. Relative to the Lowry method, the operation is simple and the sensitivity is high. However, similar to the Lowry method, it is susceptible to interference with proteins and detergents.
The Bradford method: The principle of this method is that the protein reacts with Coomassie Brilliant Blue and the resulting colored compound absorbs at 595 nm. Its greatest feature is its high sensitivity, which is twice that of the Lowry and BCA test methods; it is simpler and faster; it requires only one reagent; the compound can be stable for one hour, facilitating results; and with a series of Interfering with Lowry, the BCA reaction reducing agent (eg DTT, mercaptoethanol) is compatible. However, it is still sensitive to detergents. The main drawback is that different standards can lead to large differences in the results of the same sample and no comparable results.
Some researchers who are exposed to a colorimetric method for the first time may be confused by the results of the various colorimetric methods. Which method do you believe to believe? Because of the variety of reactions and color groups Therefore, using several methods at the same time is not comparable to the sample concentration obtained from the same sample. For example, Keller et al. tested the protein in human milk and found that the concentrations measured by Lowry and BCA were significantly higher than that of Bradford. The difference was significant. Even if the same sample is determined, the standard sample selected by the same colorimetric method is inconsistent, and the concentration after the test is also inconsistent. For example, use Lowry to test the protein in the cell homogenate, and use BSA as a standard product with a concentration of 1.34 mg/ml and a globulin as a standard with a concentration of 2.64 mg/ml. Therefore, before selecting a colorimetric method, it is preferable to refer to the chemical composition of the sample to be tested, and find a chemically similar standard protein as a standard. In addition, the colorimetric method for quantifying proteins often causes problems in that the absorbance value of the sample is too low, resulting in a large difference between the measured sample concentration and the actual concentration. The key problem is that the color after the reaction has a certain half-life, so each colorimetric method lists the reaction test time. All samples (including standard samples) must be tested within this time. If the time is too long, the absorbance value obtained becomes smaller and the converted concentration value decreases. In addition, the reaction temperature, the pH value of the solution, etc. are all important factors that affect the experiment. In addition, it is very important to use plastic colorimetry. Avoid using cuvettes made of quartz or glass because the color after reaction will cause quartz or glass to be colored, resulting in inaccurate absorbance of the sample.
Bacterial cell density (OD600)
The laboratory determines the growth density and growth period of the bacteria, and infers the growth density of bacteria based on experience and visual inspection. In the face of demanding experiments, the use of a spectrophotometer to accurately determine the bacterial cell density. OD600 is the standard method for tracking microbial growth in liquid cultures. As a blank solution, an uncultured culture solution was used, and the culture solution containing the bacteria was quantitatively cultured. To ensure correct operation, cell counts must be performed with a microscope for each microorganism and each instrument to make a calibration curve. Occasionally, the negative OD value of the broth was observed in the experiment. The reason was that a color-developing medium was used. After the bacterium was cultured for a period of time, it reacted with the medium to cause a discoloration reaction. In addition, it should be noted that the test sample cannot be centrifuged to keep the bacteria suspended.
No matter which process technology the company takes on the inner hole of the steel pipe, it can put into the inner hole of the made-up articles with the piston, gasket and flange, fitting grease outside the pipe to form a hydraulic cylinder. People in this field often called honing or cutting pipes the hydraulic steel cylinder.
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