BL A 347 General Physiology Laboratory I Calculations and Use of Spread Sheets

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1 BL A 347 General Physiology Laboratory I Calculations and Use of Spread Sheets Introduction: One of the most frequent tasks in any laboratory is the calculation of concentrations in the preparation of buffers, media, and other solutions. Whether you are a technician, pharmacist, nurse, researcher or physician, you MUST know how to accurately calculate volumes and weights for your particular applications. This first laboratory exercise is designed to either introduce you to, or refresh your memory on how to perform calculations for the laboratory or the clinic, and how to use Excel to perform many of these tasks for you. Objectives: 1. To learn that UNITS and INFORMATION are the key to any calculation. 2. To fully understand the molarity system 3. To generate a STANDARD CURVE for use in calculating unknowns. 4. To learn the basic and most frequently used aspects of Excel to generate your own spread sheets. Definitions: 1. Avagadro's number: It is generally defined as x molecules of a substance, and is the number of molecules in one mole of a substance. (You will not need to use this number in most laboratory calculations, but if forms the basis of the molarity system). 2. The gram-molecular weight (as depicted in the Periodic Table of Elements) is the weight (in grams) of one mole of a substance. For example, the formula weight (printed on the side of the bottle) of sodium choloride is This means that x molecules of NaCl weigh g. Similarly, x molecules of KCl weigh g. 2. This number is often referred to as the formula weight or molecular weight, and has units of g / mole. 3. molarity: the number of moles per liter of liquid. It is abbreviated as M. In science, most solutions are represented in units of M, usually with a prefix such as m (milli), µ (micro), etc. See list on back page of Silverthorn and below for prefix definitions. For example, a M NaCl solution is written as 25 mm, or a M NaCl solution as 250 µm.

2 Table of Prefixes deci- (d) 1/ x 10-1 centi- (c) 1/ x 10-2 milli- (m) 1/ x 10-3 micro (µ) 1/1,000, x 10-6 nano- (n) 1/1,000,000, x 10-9 pico- (p) 1/1,000,000,000, x kilo- (k) 1,000 1 x x 10 3 "Rule of 3" OK, now let's get down to business and do a step-by-step example of how to prepare solutions for the laboratory. Example 1: You are performing a liver perfusion experiment and you need 500 ml of an isotonic saline solution. The final concentrations of each of the components are: NaCl (58.44), 140 mm KCl (74.55), 8 mm Glucose (180.2), 5 mm What weight of each do you need to add to the solution to achieve these final values? Step 1: Look at the information you are given: formula weights & volume (0.5 liters). Step 2: Use the following "magical formula" to calculate the values: g of substance = formula weight (g/mole) X desired concentration (moles/liter) X volume (liters) g = FW x M x V (Equation 1) NOTICE HOW THE UNITS CANCEL AND GIVE YOU THE DESIRED RESULT: g/mole x moles/liter = g/liter x liters = g Remember this formula, because you can calculate any solution preparation with it. Let's solve the problem: NaCl = x M x 0.5 liters = 4.09 g KCl = x M x 0.5 liters = g (or 298 mg) Glucose = x M x 0.5 liters = g (or 451 mg) Example 2: You just obtained a very expensive reagent to perform a vital experiment on the role of phosphorylation in a signal transduction pathway that you study in kidney cells. The reagent, Okadaic acid (FW = 805) is a phosphatase inhibitor produced by a marine sponge species, and it arrived from the supplier with a "spec sheet" that suggests you reconstitute the 10 µg of lyophilized product in 1 ml of dimethylsulfoxide (DMSO) for your STOCK SOLUTION. You know that you need to use it at a concentration of 1 nm. What volume of the reconstituted stock solution should you add to 50 ml of tissue culture medium to use in the experiment? Solution: Let's look at our information and objective: Okadaic acid FW = 805, quantity = 10 µg, volume DMSO = 1 ml And we need: A final volume of 50 ml with a 1 nm final Okadaic acid concentration.

3 First, we need to establish the molarity of our stock solution (Molarity = moles/liter). g = FW x M x V rearranged, we obtain M = g/fw/v Therefore, 1 x 10-5 g 805 g/mole = 1.24 x10-8 moles liters = 1.24 x 10-5 M Second, we need to know how much (volume) of this solution to add to the 50 ml of media to achieve the final desired concentration of 1 nm. This is just a simple ratio, since they have the same units: stock concentration desired concentration tells you how many times more concentrated your stock solution is compared to your desired final concentration. In this case, it's 1.24 x 10-5 M 1 x 10-9 M = 1.24 x 10 4 X more concentrated, or in other words, we need to dilute it 1.24 x 10 4 X. How much do we add? 50 ml 1.24 x 10 4 = 4.03 x 10-3 ml or 4.03 µl. *For most researchers, it is easier to make a 1,000X stock of a solution, so that you can simply add 1 µl per ml of solution. What volume of DMSO would have to have been added to the Okadaic acid to achieve this from the outset? A1,000X stock for 1 nm would require a 1 µm solution: 1 x 10-5 g 805 g/mole = 1.24 x10-8 moles 1 x 10-6 moles/liter = liters or 12.4 ml Calculation of rates Remember, rates are always expressed per unit time. For example: Flow rate = ml/min, Metabolic rate = kcal/kg/day Cellular nutrient transport rates = nmoles/mg protein/min External respiration rates = ml air/min Example 3: A patient comes in with multiple embolisms in his lower extremities. The doctor immediately orders an i.v. heparin drip for the patient to prevent further clotting or formation of pulmonary embolisms. The heparin bag is supplied at 250,000 units in 250 ml, and the specific activity of heparin is 10,000 units/mg. The doctor states that he wants the patient to receive 10 mg of heparin per day. What should the flow rate of the metering pump be set at, in ml/min to achieve this delivery rate? (hint: conversions and units of measure). Again, the FIRST step is to assess the information and apply it to what is being asked. Heparin is supplied at 1,000 units/ml, and the specific activity of heparin is 10,000 units/mg. Since the doctor wants heparin delivered in terms of mg, we must convert the concentration to mg/ml: 1,000 units/ml 10,000 units/mg = 0.1 mg heparin/ml. Armed with this information, we can calculate the volume of heparin solution to be infused: 10 mg 0.1 mg/ml = 100 ml Now we come to the rate: we need 100 ml/day, but the units on the pump are in ml/min. Simply convert days to min: 100 ml/day x 1 day/24 hr x 1 hr/60 min = 100 ml/1440 min or ml/min.

4 Calculating values for experimental samples from Standard Curves Researchers and clinicians frequently need to quantify values of experimental samples based on photometric analyses (spectrophotometry, fluorescence, scintillation spectrophotometry, etc.). To achieve this, they first measure samples with known values and generate standard curves. Based on the relationship between the absorbance and concentration (the slope of the line), they can calculate values for their experimental (unknown) samples. Today, you will generate a standard curve for protein using the bicinchoninic acid (BCA) procedure. You will then graph your data, calculate the slope of the line, and use the resulting equation to calculate values for unknowns in Excel. Instructions: Protein Standard Curve 1. Label each of 5 microfuge tubes as: , 0.125, and 0.50, and place them in the rack. 2. Using the pipetman, place 200 µl of H 2 O into each of the 5 tubes from the beaker at your station. 3. From the tube labeled as "1" in your rack, draw up 200 µl of this 1 mg/ml protein solution and place into the tube labeled 0.5. Vortex tube on electronic mixer, and transfer 200 µl of solution to tube labeled Repeat procedure until you have placed 200 µl of solution into tube labeled This is simply a serial two-fold dilution of protein solutions that you will use in your standard curve. 4. Starting with 10 µl of H 2 O in the first well, place 10 µl of each of your solutions in horizontally adjacent wells of the 96-well plate. You should have 6 wells occupied. 5. Now, place 200 µl of BCA reagent in each of the six wells. We will be using one 96-well plate per table, so make a note of which set of wells are yours. 6. Read samples in the 96-well plate reader at a wavelength of 562 nm. 7. Record the absorbances (A 562 ) in an Excel data sheet. 8. Highlight and graph your data using the chart wizard; choose Scatter diagram. 9. Follow the menu commands and click "Finish" when you're done. Click on data points in graph; under "Chart" in the upper menu, choose "Add Trendline" and then choose the linear model in the box. 10. Click on the Options tab in the box and check the boxes that say "display equation on chart" and "display r-squared value on chart".

5 11. The equation gives you the slope of your line and the r 2 value gives you the quality of your curve, with a value of 1.0 as perfect. 12. Use this equation in Excel to calculate your unknown samples below. Use your standard curve slope to calculate the following unknown protein concentrations: A Protein concentration (mg/ml) Blank (10µl of H 2 O) Order: 0, , 0.125,0.25, 0.5, 1.0 Place your samples in any of these rows.