Biology 3T: Week 1-2
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This 5 page Class Notes was uploaded by Tiffany Thomas on Tuesday January 12, 2016. The Class Notes belongs to 01202 at Rowan University taught by Dr. Grove in Fall 2015. Since its upload, it has received 110 views.
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Date Created: 01/12/16
Biology 3T Reagent preparation instructions Basic lab protocol 1. Safety first! Never eat or drink in the lab. Know if the materials that you are using are hazardous read the label and refer to the material safety data sheet (MSDS) which are available from the manufacturer if necessary. Wear gloves, lab coats, eye protection, and masks if necessary. 2. Keep the lab free of clutter and clean up after yourself. All dishes must be washed with soap and thoroughly rinsed. Rinse each piece of glassware at least 5 times (3 times with tap water, 2 times with distilled water). Hang on drying rack until dry. 3. Use micropipettors carefully. Pipettors are very expensive. Be sure that you know how to use them properly. Helpful information a molar (M) solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight. (e.g. 1M = 1 mole/L) o Example: the molecular weight (MW) of NaCl is 58.44 g/mole so, a 1 M NaCl solution would have 58.44 g / L of solvent percentage (weight/volume) = # g /100 mL of solvent percentage (volume/volume) = # mL / 100 mL of solvent milli micro nanno pico femto 3 6 9 12 15 10 10 10 10 10 molar units mM M nM pM fM volume units mL L nL pL fL There are two basic types of calculations 1) How much of each reagent do you add to make up a solution? Example: make up 100 mL of a 5M NaCl solution Use the equation: molecular weight (g/mole) * # moles/L * volume needed (in L) So: 58.44 g/mole * 5 moles/L * 0.1 L = 29.22 g NaCl Then: add distilled water (d2 0) to bring the concentration up to a final volume of 100 mL Reagent preparation instructions 1 Note: always pay attention to units!! Make sure your units are consistent in the calculations. For example, above, convert 100 mL to 0.1 L because you’re also using molarity, in moles/L 2) How do you use an existing solution at a known concentration, to make a new solution at a new concentration? Example: you have a stock solution at 100 g/L, and you need 100 mL of a solution at 1 g/L. How much of the stock solution do you use? How much distilled water do you have to use? Use the equation: C1V1 = C 2 2 where C = concentration and V=volume o Here’s what you have: stock solution with a known concentration (C = 100 1 g/L). o Here’s what you need: a particular volume (V = 1002mL = 0.1 L), at a particular concentration (C =21 g/L) o To find the volume of the stock solution to use (V ): 1 C V = C V 1 1 2 2 (100 g/L)(V )1 = (1 g/L)(0.1 L) V1 = 0.001 L = 1 mL of the stock solution Note: You’re not done yet! You have now calculated the volume of the stock solution you need. But you wanted a total of 100 mL at 1 g/L. You’ve taken 1 mL of the stock solution, and now you will need to add distilled water (dH O) to 2ring the final volume up to 100 mL (so 99 mL dH 0). 2 Making the solutions A. Write down all of your calculations and actual steps taken in making the solution, including units on all numbers, in your lab notebook. B. Obtain clean glassware. C. Weigh out the desired amount of chemicals using weigh paper or a weigh boat. D. Place chemicals in an appropriate sized beaker with a stir bar. E. Add less than the required final amount of water. A general rule is to start with 8090% of the final volume. Prepare all solutions with distilled water (dH O); 2ap water is for washing your hands and rinsing your dishes only. F. Place on a stir plate, with a spinning stir bar, until dissolved. Reagent preparation instructions 2 G. Test the pH. If it’s not at the desired level, adjust the pH using HCl (↓) or NaOH (↑). H. Transfer the solution to a graduated cylinder. Add the required amount of distilled water to achieve the final volume. Reagent preparation instructions 3 Some practice problems 1) Make a 100 mL solution of 1M TrisHCl and 0.15M NaCL at a pH of 7.5. Molecular weights: TrisHCl = 157.6 g/mol; NaCl = 58.44 g/mol). Do the calculations for how much of each reagent you would use, then describe the steps to mix up this solution, as outlined above under “Making the solutions” 2) You have 5M NaCl stock and you need 100mL of 0.15M NaCl. How do you make this solution? 3) Calculate how many grams of glucose would be required to prepare 400 mL of a 0.36M solution. Include units on your calculations. Molecular weight of glucose = 180 g/mole. 4) You have a 100X stock of a buffer. You need 250 mL of 3.5X buffer. How much of the stock solution do you use? How much distilled water (dH O)2 5) Arrange the following units of molarity into the correct order from largest to smallest. pM, fM, mM, M, nM 6) Make 500 mL of the following solution: 10 mM TrisHCl; 0.25 M Glucose; 0.1% Tween20; 5% BSA, at a pH of 7.5. (Hint: calculate each reagent in this solution separately, then figure out how much dH O you’d 2 need to add to get to the final volume. Write out the steps as shown above, under “Making the solutions”) Information you’ll need: Molecular weight of TrisHCl = 157.6 g/mole. You have a 3M glucose stock. Tween20 is a liquid. BSA is a solid. Questions: How many grams of TrisHCl do you use? How much of the 3M glucose stock do you use? How much Tween20 do you use? How much BSA do you use? Describe the process of making the solution (follow the recipe in “Making the solutions” shown above) Show all your calculations. Be sure to put units on every number Reagent preparation instructions 4 Calculations for Lab 1 ** All of these calculations should go in your laboratory notebook. If you don’t have one yet, tape or staple these calculations into the lab notebook once you purchase one. 1a) Do the calculations to mix up the following solution for our lab next week: Yeast growing medium: 100mL of 56 mM glucose, 20 mM HEPES at pH 6.8. Information you may need glucose molecular weight = 180.16 g/mole HEPES molecular weight = 260.30 g/mole Molarity (M) is in moles / L Remember units as you do your calculations (e.g. what is 56mM, expressed in M?), and always put units after every number!! 1b) Write out how you would mix up this yeast growing medium solution, step by step, as is shown above under “Making the solutions” 2) Do the calculation to make a 10 mL stock solution of 1.0% neutral red dye. How much dye and how much yeast growing medium (YGM) you need? Information you may need Neutral red dye comes in a powder (i.e. it’s a solid) 3) Using your neutral red dye stock solution (at 1%), do the calculations to mix up 1 mL of each of the following concentrations: 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0%. Reagent preparation instructions 5
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