Cuvettes cleaning

Never clean cuvettes or any optically polished glassware with ethanolic KOH or other strong base, as this will cause etching. All cuvettes should be cleaned carefully with 0.5% detergent solution, in a sonicator bath, or in a cuvette washer. 

Calibrated fluorescence spectrometer, buffer solution, and cleaned cuvettes (see Strategic Planning)... 

Fill a cleaned cuvette with a filtered sample from the actual batch of buffer used to dissolve or dialyze the protein, then place the cuvette in the cell holder of the spectrometer in a reproducible orientation (see Strategic Planning, discussion of Cells). Scan this buffer blank using the same instrument settings as are appropriate for the sample, store the spectrum, and check for any unexpected fluorescence bands. 

An absorbance measurement of a sample is obtained as follows. Rinse a clean cuvette or the indexed sample test tube with distilled water and then with several milliliters of sample solution. Half fill the cuvette or the test tube with sample solution, and make certain that the solution is free of bubbles and that the exterior surface of the cuvette or the test tube is wiped clean with a tissue. Then insert the cuvette or the test tube carefully into the sample holder, and close the door. Read the absorbance of that solution directly from the meter. Then remove the cuvette or the test tube from the sample holder, and rinse the cuvette or the test tube with distilled water so that it is ready to receive the next sample. Repeat the above procedure until all absorbance readings at a given wavelength have been obtained. 

Prior to describing the various mechanisms of sensing, we consider it usefiii to expand on the use of decay-lime measurements for sensing. The advantages of lifetime-based sensing are illustrated in Rgure 19.5. In the research laboratory, where clean cuvettes and i cal sutfeces are... 

The importance of clean cuvettes is self-evident. Routinely, all non-disposable cuvettes should be emptied immediately after use, rinsed repeatedly in the solvent (e.g. water), then with clean ethanol or acetone and dried with low pressure air or nitrogen from a cylinder. It is prudent to install a filter (such as those with pore sizes of 0.45 pm used in filter sterilization) in the gas line. Cuvette washers (e.g. Aldrich) wash, rinse, and dry cuvettes. Cotton wool buds can also be usefiil for dislodging interior, stubborn marks and for drying. The outside optical surfoces should be polished with clean lens tissue. Note that plastic squeety bottles generally used for solvents contain plasticizers such as butyl phthalate, which can interfere with critical UV spectra. 

Cuvette material Optically clean glass Electrode cell material Organic glass... 

Cuvettes used with the spectrometer are not simply test tubes. They are specially made tubes or cuvettes and are often matched such that a set of tubes or cuvettes will all have the same absorbance characteristics. Cuvettes should never be used as test tubes they must be kept clean at all times, and care must be taken not to scratch them. When using cuvettes that have not been used before, they should be tested to make sure they are all the same. This is accomplished by inserting them into the spectrophotometer and noting their absorbance. All should be the same. Keep in mind that empty cuvettes will have a higher absorbance than when filled with water. This is because light is refracted at each surface, and when filled with water or solvent, there is less refraction at the surfaces. 

Fill a cuvette about three-fourths full with distilled water and dry its outside with a tissue. To calibrate the colorimeter, place the cuvette in the colorimeter and close the lid. Turn the wavelength knob to 0%T. Press TRIGGER on the CBL and enter 0 into the calculator. Turn the wavelength knob to Red (635 nm). Press TRIGGER on the CBL and enter 100 into the calculator. Leave the colorimeter set on Red for the rest of the lab. Remove the cuvette from the colorimeter. Empty the distilled water from the cuvette. Dry the inside of the cuvette with a clean cotton swab. 

Remove the cuvette and pour out the solution. Rinse the inside of the cuvette with distilled water and dry it with a clean cotton swab. Repeat this step for test tubes 2 through 5. 

Clean the cuvette with a cotton swab and fill it about three-fourths full with the unknown dye solution. Place the cuvette in the colorimeter and close the lid. From the MAIN MENU, select COLLECT DATA (do not select SET UP PROBES as this will erase your data lists). Select MONITOR INPUT from the DATA COLLECTION MENU. Press ENTER to monitor the absorbance value of the colorimeter. After about 10-15 seconds, record the absorbance value and record it in your data table. 

Turn off the colorimeter. Clean and dry the cuvette. Return all equipment to its proper place. 

Remove the sample, clean the cuvette and fill with another sample for readings. 

Practical Considerations. Typical absorption assay methods utilize ultraviolet (UV) or visible (vis) wavelengths. With most spectrophotometers, the measured absorbance should be less than 1.2 to obtain a strictly linear relationship (/.c., to obey the Beer-Lambert Law). Nonlinear A versus c plots can result from micelle formation, sample turbidity, the presence of stray light (see below), bubble formation, stacking of aromatic chromophores, and even the presence of fine cotton strands from tissue used to clean the faces of cuvettes. One is well advised to confirm the linearity of absorbance with respect to product (or substrate) concentration under the exact assay conditions to be employed in... 

In order to determine the specific activity of the enzyme, the exact concentration of the enzyme must be known. The concentration of the solution of tyrosinase may be determined as a class project by the following procedure. Turn on the spectrophotometer and the UV lamp. Adjust the wavelength to 280 nm. Allow the instrument and lamp to warm up for 15 to 20 minutes. Transfer 1.0 or 3.0 mL of the phosphate buffer to a 1- or 3-mL quartz cuvette. Place it in the sample position of the spectrophotometer and adjust the balance to zero absorbance. Discard the buffer, and clean and dry the cuvette. Transfer 1.0 or 3.0 mL of the tyrosinase solution into the quartz cuvette. Place in the sample position and record the absorbance at 280 nm. Calculate the tyrosinase concentration as described in the Analysis of Results section. 

Pour the contents of the cuvette into the waste ethidium container provided by your instructor. Clean the cuvette carefully with warm water, rinse several times with distilled water, and dry. 

Standardize the spectrofluorimeter in the following way. Pipet 2.0 mL of the pH 7.5, ethidium bromide-Tris buffer into a cuvette. Add 1.0 mL of Tris buffer I and 20 fiL of standard DNA solution. Mix and place in the fluorimeter. Adjust the fluorescence intensity to 100. Clean the cuvette as described in part A and repeat the assay using various concentrations of spermine. Prepare a table displaying the amount of each component to be added. Four reagents must be in the table pH 7.5, ethidium bromide-Tris buffer, DNA solution, spermine, and Tris buffer I. Maintain the volume of DNA at 20 fiL and ethidium bromide solution at 2.0 mL for all assays. Use 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of spermine in the assays. Remember that the total volume of all constituents in the cuvette must remain constant at 3.02 mL for all the assays. Therefore, the amount of Tris buffer I must change with the amount of spermine added. Prepare each assay separately by adding the proper amount of each component to the cuvette. Mix well and record the fluorescence intensity of each cuvette. 

Disposable polystyrene cuvettes eliminate thejob of cleaning dye-stained quartz or glass cuvettes. 

A single spectrum, with associated baseline, will require -1 hr, including the time needed to fill and clean the cuvette. Up to an additional hour may be needed for optimizing instrument parameters if the sample is unfamiliar. For a number of samples, economies of time can be effected (see Basic Protocol). 

Make sure that cuvette surfaces are clean. 

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