Materials: Micropipettes (2-20 µl range pipette, µl range, µl range), tips, test tubes with color dye, well plates
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1 Virtually every chemical reaction in a lab or manufacturing facility, as in cells, occurs in a watery environment or solution. A lab technician, therefore, must be able to quickly prepare any volume of solution at any concentration of molecules. Solution preparation is the most basic laboratory skill required of every scientist or technician. Solution prep involves measuring liquid volumes with a variety of instruments, weighing chemicals with a balance or scale, mixing them together in the correct proportions. Lab 1: Using a Micropipette Background: Very tiny amounts of chemicals and biological reagents are used in many biotechnology experiments. To measure these minute volumes, technicians use micropipettes that measure microliter (µl) amounts. Materials: Micropipettes (2-20 µl range pipette, µl range, µl range), tips, test tubes with color dye, well plates Things to remember Never use the micropipette without a tip. Never lay a loaded micropipette down or tilt it as this could allow liquid to run into the pipette. Never allow the button to snap back into place. Always allow the button to slowly return to the up position. Never adjust the micropipette beyond its measurable range. 1. Set the dial at the correct measurement using the green knob. 2. Put the tip on the end of the pipette. 3. Depress the plunger to the 1 st stop position. Hold the micropipette vertically; put the tip into the colored liquid. 4. Release the pressure allowing the plunger to move up slowly. This will draw the liquid up into the tip. There should be no bubbles in the liquid in the tip if you have done this correctly. 5. Withdraw the tip from the liquid & squirt the liquid into the correct well by pushing the plunger to the 2 nd stop position. 6. With the plunger still depressed, withdraw the tip from the well. Allow the plunger to slowly rise to the up position. 7. Remove the tip by pressing on the eject button. Put the used tips in an empty beaker. Continue adding the appropriate amount to the wells. 1
2 Cell Yellow Red Blue Green Cell Yellow Red Blue Green D µl 0 C7 50 µl 50 µl 50 µl 200 µl E µl 0 D7 50 µl 50 µl 50 µl 200 µl F µl 0 F7 50 µl 50 µl 50 µl 200 µl C8.2 ml 0 50 µl 100 µl C6.1 ml 50 µl 50 µl 150 µl F8.2 ml 0 50 µl 100 µl E6.1 ml 50 µl 50 µl 150 µl A µl 25 µl 0 0 F6 25 µl 50 µl 50 µl 150 µl G µl 25 µl 0 0 C5 25 µl 100 µl 225 µl 0 B µl 100 µl 0 25 µl F µl 225 µl 0 C µl 100 µl 0 25 µl C µl 50 µl D µl 100 µl 0 25 µl F µl 50 µl E µl 100 µl 0 25 µl C µl 250 µl 25 µl F µl 100 µl 0 25 µl F µl 250 µl 25 µl C10 75 µl.25 ml 0 25 µl D µl 250 µl 0 E10 75 µl.25 ml 0 25 µl E µl 250 µl 0 D µl 0 0 F µl 250 µl 0 Lab 2: Mass/Volume Equivalents A balance can be used to determine if a micropipette is measuring within an acceptable range. Since 1 ml of water weights 1.0 g, you can estimate the expected mass for any volume of water. Water dispensed by a micropipette can be weighed on a balance. By comparing the actual observed mass to the expected mass, you can make an error determination. (observed mass expected mass) Expected mass x 100 = % error determination Materials: Micropipettes, tips, tap water, scale Data: Volume Volume Expected Actual Acceptable µl ml mass g mass g % Error Error % 100 µl 5 % 50 µl 5 % 20 µl 5 % 10 µl 3% For any error that is outside of the acceptable range, re-measure. Make sure that you are using the instruments correctly. If you still get measurements outside the range of acceptable error, the pipet may be out of calibration. 2
3 LAB 3: Making Solutions of Differing Mass/Volume Concentrations Solutions are prepared with a certain mass of solute in a certain volume of solvent. Although concentrations can be reported in any mass/volume units, these units are the most common in biotechnology applications. g/ml grams per milliliter µg/µl micrograms per microliter g/l grams per liter ng/l nanograms per liter mg/ml milligrams per milliliter ng/µl nanogrmas per microliter µg/ml micrograms per milliliter To determine how to prepare a certain volume of a solution at a certain mass/volume concentration, use the equation. concentration desired x total volume desired = mass of solute in total volume desired Ex. Suppose a technician needs 50 ml of 15 mg/ml pepsin solution. What is the mass of the solute in the total volume desired? 50 ml x 15 mg/ml = 750 mg =.75 g pepsin Materials: scale, weigh boat, lab scoops, test tubes, tube racks, deionized water, pipets, sugar 1. Label all test tubes with the sample name and concentration, your initials and the date. 2. Prepare the solutions listed in the table in test tubes using deionized water as the solvent. Use the mass/volume concentration equation to determine mass of sugar to use. Tube # Glucose solution to be made 1 5 ml of 200 mg/ml 2 10 ml of 200 mg/ml 3 15 ml of 200 mg/ml 4 20 ml of 200 mg/ml Data Table: Tube # Total Volume ml Concentration mg/ml Mass of Solute Used g Test Strip Results mg/dl 3. Determine the concentration of the prepared samples by matching the color of the test strips to the standard concentration key on the test strip package. Record the test strip data for each sample. 3
4 Lab 4: Making Solutions of Differing % Mass/Volume Concentrations A lab technician must be able to make any solution at any concentration or volume. Technicians must be able to recognize which chemicals should be measured out, in what amounts, and what math must be done to calculate these amounts. Use the % mass/volume concentration equation to determine grams of solute needed. Example: To make 40 ml of 5% NaCl solution. Step 1: Convert the % to a decimal. % = g/ml (decimal value) Example: 5% = 5 g/ml Divide the % by 100. Step 2: Use the mass/volume equation. Ex. 5g/100 ml = x/40 ml x = 2 g of NaCl o Add solvent to solute until the desired volume is reached. Materials: scale, weigh boats, lab scoops, test tubes, tube racks, gelatin 1. Calculate the amount of gelatin needed to make each gelatin solution. Show your calculations. 2. Use the scale to weight out the gelatin samples. 3. Pour each gelatin sample into the appropriately labeled test tubes. 4. Slowly add dh 2 0 to bring the mixture to a total volume of 5 ml in each tube. 5. Add 10 drops of Biuret Reagent to test for the presence of a protein. Data: Tube Label Mass of solute used g Total volume ml Color 6% gelatin 5 ml 3% gelatin 5 ml 1% gelatin 5 ml 0% gelatin 5 ml Analysis: Describe the results of the Biuret testing of the gelatin solutions of decreasing concentrations. 4
5 Lab 5: Making Solutions of Differing Molar Concentrations The concentration of many solutions is reported as moles/liter (mol/l or M - molar). The concentration measurement is called molarity. Use the molarity concentration equation to determine the grams of solute needed. MW = molecular weight Volume wanted x molarity desired x MW of solute = grams of solute to be dissolved (L) (mol/l) (g/mol) in solvent to the final desired volume Materials: scale, weigh boats, sugar, deionized water, lab scoops, 250 ml beakers 1. In 5 labeled beakers, prepare the solutions using deionized water as the solvent. Use the Molarity Concentration Equation to determine the mass of sugar to use in each solution. Beaker # Total Volume (ml) Concentration (M) ml.2 M ml.4 M ml.6 M ml.8 M ml 1 M Mass of Solute Used (g) Calculations: 5
6 Lab 6: Making Dilutions of Concentrated Solutions Making dilutions of concentrated solutions is a common practice in biotechnology lab. A concentrated solution is generally called a stock solution, and the diluted solution is called working solution. Preparing a concentrated stock solution saves a lot of time and is easier to store than large volumes of diluted working solutions. Making a working solution requires diluting some volume of stock solution to the concentration needed. Example: A technician has a stock bottle of 1M TRIS solution but needs 150 ml of.1 M TRIS for an experiment. Use the dilution equation. C 1 V 1 = C 2 V 2 (1M)(V 1 ) = (.1M)(150mL) V 1 = 15 ml C 1 = the concentration of the concentrated stock solution (starting solution) V 1 = volume to use of the stock solution in the diluted sample C 2 = desired concentration of the diluted sample V 2 = desired volume of the diluted sample Materials: graduated cylinder, deionized water, 500 ml Erlenmeyer flask, 50X TAE buffer 1. Make 100 ml of a 1x TAE buffer Starting stock concentration (X) Volume of concentrated stock needed (ml) Dilution concentration needed (X) Diluted volume desired (ml) 50 X 1X 100 ml Lab 7: Making Serial Dilutions When a number of dilutions must be made and each is proportionally the same dilution as the one before, the process is called a serial dilution. A serial dilution is useful for preparing dilute solutions that are hard to make from scratch, because the solute can be too small to measure. Materials: sugar, deionized water, scale, weigh boats, lab scoops, 1000 ml beaker (3) 1. Label 1000 ml beaker -- 1M NaCl. 2. Make 1000 ml of a 1M NaCl solution. Use the formula to calculate the amount of solute to add. Volume wanted x molarity desired x MW of solute = grams of solute to be dissolved (L) (mol/l) (g/mol) in solvent to the final desired volume 3. Label ml beakers.5 M and.25 M 4. Solution A: 1000 ml solution of.5 M a. Pour 500 ml of 1M solution into beaker. Add 500 ml of deionized water. 5. Solution B: 1000 ml solution of.25 M a. Pour 500 ml of.5 M solution into beaker. Add 500 ml of deionized water. 6
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