Lab 4: Molecules in Biotechnology; Ph & Buffers; DNA Isolation From

Lab 4: Molecules in Biotechnology; pH & Buffers; DNA Isolation from Strawberries

We will begin today’s lab with a lecture on cellular molecules in biotechnology. There are four main categories of cellular molecules:

1) Carbohydrates 2) Lipids 3) Proteins 4) Nucleic acids

The lecture is in Powerpoint format, and I have printed the slides for you so that you can follow along and take additional notes. From this point on in the semester, be aware of each reagent that we use in lab and know what category of cellular molecule it falls into. Each category of cellular molecule has different biological and chemical properties, so it is always helpful to know the type of molecule with which you are working during an experiment.

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Activity 4a

Measuring the pH of Solutions

Purpose In this activity you will learn about pH and test the pH of some common solutions. The pH of a solution can be extremely critical to its biological and chemical properties. Solvents of a specific pH are often necessary in biotechnology labs to prepare solutions of DNA, protein, nucleic acids, and other cellular molecules.

Background

Definition of pH: pH is a measurement of the concentration of hydrogen ions (= H+, or protons) in a solution. Numerically pH is defined as the negative logarithm of the concentration of H+ ions expressed in moles per liter (M). pH = -log [H+] Pure water spontaneously dissociates into ions, forming a 10-7 M solution of H+ (and OH-). The negative of this logarithm is 7, so the pH of pure water is 7. Solutions with a higher concentration of H+ than occurs in pure water have pH values below 7 and are acidic. Solutions containing molecules or ions that reduce the concentration of H+ below that of pure water have pH values above 7 and are basic or alkaline. neutral acidic basic

p 0 ic 7 1

H 4

[H+] in 0 -7 1x10 1x10 1x10-14 mol/L (as in pure water)

Because the pH scale is a logarithmic scale, each pH unit represents a 10-fold change in the concentration of H+ ions.

Some questions for practice: 1. Lemon juice has a pH of 2. Tomato juice has a pH of 4. a) Which type of juice is more acidic?

b) How many times more acidic?

2) You have two solutions of the same volume. One has a pH of 7, and one has a pH of 11. a) Are these solutions acidic, neutral, or basic?

b) Fill in the blanks in the following sentence. The solution with pH 7 has times more H+ ions than the solution with pH 11 .

39 Why is pH important in biology? Most biological molecules, including cellular molecules such as proteins, nucleic acids, carbohydrates, and lipids, are sensitive to changes in pH. If there are drastic changes in pH, these molecules won’t function properly. The optimal pH range for most living things is between 6.5 to 8.2.

Here are some other examples of the importance of pH (adapted from http://www.ph.co.za/):

1. Drinking water purification depends on correct pH for its operation.

2. In sugar manufacture, improper pH can result in formation of unwanted acids and very little sugar.

3. In sewage treatment, pH must be adjusted for proper operation of disposal plants. This is, of course, another reason why polluting sewers with high acid or alkaline material spills is undesirable.

4. Milk turns sour at a pH of 6.00. Thus good milk requires good pH control.

5. In old-fashioned jelly making the pH must be < 4.0, or the jelly will not jell.

6. The brightness of chrome coating on our auto bumpers is directly related to the pH of the plating solution.

To sum up, almost every manufacturing process that involves even the simplest chemical reaction is sensitive to pH and will usually produce best results at some optimum pH value. One of the key factors in keeping consistent, uniform products is the ability to measure and maintain pH at the proper level.

Procedure for Measuring pH You will measure and record the pH of a number of different household solutions using two different methods: pH indicator strips and a pH meter. pH indicator strips are much quicker to use, but less sensitive and usually less precise than a pH meter. Our pH indicator has gradations of 0.5 pH units. Therefore, a pH of 6.8 will probably read as 7.0 with pH paper, whereas a pH meter (if calibrated correctly) will give a more sensitive reading closer to the actual pH of 6.8. pH indicator strips 1. These strips can be narrow-range or broad-range. Ours are broad-range (meaning that they cover most or all of the pH scale from 0 – 14). Each strip has 8 chemical indicators that change color with different pH. 2. Dip the pH indicator strip into the solution to be tested and immediately remove the strip and lay it on a paper towel indicator side up. Compare the colors on the strip to the colored key on the box. pH meter 3. The pH meter uses an electrode that can measure the [H+] very sensitively. Each day or so, the pH meter must be calibrated with buffer standards so that it gives accurate pH measurements. 4. Before using the pH meter, your instructor will give you a demonstration of proper pH meter procedure. It is very important that you are careful with the glass electrode to avoid breaking it. Also, you must rinse the electrode with deionized water before and after each reading so

40 as not to contaminate the different solutions. Use your squirt bottle and a waste beaker to do this. 5. Create a data table similar to Table 4.1 below to record your measurements. Are the two pH measurements for each solution similar? Are your pH values for each solution similar to that obtained by other groups?

Table 4.1 pH Measurements of Solutions

Solution pH (pH paper) pH (pH meter)

6. Draw a pH line and label it from 0 to 14 to report the relative pH value of each solution. Each solution tested should be added to the pH line. Use the pH determined by the method in which you have the most confidence, either pH paper indicator strips of a pH meter.

Activity 4b Making a Buffer Solution of Specific pH

Purpose Different biological molecules require different solvents of a specific pH in order to function optimally. In this activity you will make a buffer solution containing two ingredients (together, in one solution) that has a pH of 7.2. You will use your buffer in a couple of weeks to make an enzyme solution.

Background The definition of a buffer is: a solution that resists changes in pH. This means that if an acid or base is added to a solution containing a buffer, the pH won’t change much; in contrast, if there is no buffer present in the solution, any addition of acid or base would cause a drastic change in the pH of the solution. Some commonly used buffers in biology are Tris buffers and phosphate

(PO4)-based buffers.

Procedure: 1. You will be making 50 mL of the following buffer:

50 mM Tris, 5 mM CaCl2, pH 7.2

2. First, calculate the appropriate amounts of Tris (FW = 121) and CaCl2 (FW = 111) to weigh. You may want to check your calculations with your instructor before proceeding.

41 3. Mix the Tris and CaCl2 in a 50 mL plastic conical tube with 25 mL deionized water until dissolved (you can cap the tube and shake or vortex to dissolve the Tris and CaCl2. 4. Check pH with indicator strips or pH meter. 5. Adjust the pH by adding either 1M HCl or 1M NaOH until the pH is 7.2. (Note: use caution when handling HCl (strong acid) and NaOH (strong base); wear gloves and goggles). 6. Once the pH is at 7.2, add enough deionized water to bring the total volume up to 50mL. 7. Mix thoroughly, then check pH again to make sure it’s still at 7.2. 8. Label your buffer tube with the concentrations, pH, your group’s initials, and the date, and store your buffer at 4° C in the rack indicated by your instructor.

Activity 4c Demonstration of Buffer Efficacy

Purpose In this activity you test the efficacy of two different buffers by measuring the pH as you add increasing amounts of either strong acid (HCl) or strong base (NaOH). Each buffer will be compared with deionized water in order to easily discern the effect of the buffer in resisting changes in pH.

Procedure 1. You will be using two different buffers: a 0.1 M Tris buffer at pH 8.0 and a 0.1 M sodium

monophosphate (NaH2PO4) buffer at pH 4.5. I will prepare the buffers for you in order to save time, but you should do the calculations necessary to prepare 50 mL of each buffer in your lab notebook. You can check your calculations with the instructor before proceeding.

2. First, measure and record the buffering efficacy of water. Table 4.2 d.i. H2O a) Add 25 mL of d.i.H2O to a small beaker. Vol. HCl pH b) Measure the pH of the d.i.H2O with a pH meter. added c) Add 20 µl of 1 M HCl to the d.i.H2O (use caution! Gloves & 0 µl goggles required). 20 µl d) Mix well. Determine the new pH of the water solution. 40 µl e) Repeat steps c and d four times, for a total of 100 µl of acid 60 µl added. Record all the data in a table like Table 4.2. 80 µl f) Now, rinse out the beaker well, and add 25 mL of fresh d.i.H O. 2 100 µl g) Measure the pH of the d.i.H2O with a pH meter.

h) Add 20 µl of 1 M NaOH to the d.i.H2O (use caution! Table 4.3 d.i. H2O Gloves & goggles required). Vol. NaOH pH i) Mix well. Determine the new pH of the water solution. added j) Repeat steps h and i four times, for a total of 100 µl of base 0 µl added. 20 µl k) Record all the data in your notebook in a table like Table 4.3. 40 µl 60 µl

80 µl

100 µl

42 3. Next, measure the buffering efficacy of the 0.1 M NaH2PO4 pH 4.5 buffer. Table 4.4 NaH2PO4 a) Add 25 mL of 0.1 M NaH PO pH 4.5 buffer to a clean small beaker. 2 4 Vol. NaOH b) Measure the pH of the NaH PO buffer. pH 2 4 added c) Add 20 µl of NaOH to the buffer (use caution! 0 µl Gloves & goggles required). d) Mix well. Determine the new pH of the buffer solution. 20 µl e) Repeat steps c and d four times, for a total of 100 µl of base 40 µl added. 60 µl f) Record all the data in your notebook in a table like Table 4.4. 80 µl 100 µl

4. Next, measure the buffering efficacy of the 0.1 M Tris pH 8 buffer. Table 4.5 Tris Vol. HCl a) Add 25 mL of 0.1 M Tris pH 8 buffer to a clean small beaker. pH b) Measure the pH of the Tris pH 8 buffer. added c) Add 20 µl of HCl to the buffer (use caution! 0 µl Gloves & goggles required). 20 µl d) Mix well. Determine the new pH of the buffer solution. 40 µl e) Repeat steps c and d four times, for a total of 100 µl of acid 60 µl added. 80 µl f) Record all the data in your notebook in a table like Table 4.5. 100 µl

5. Data analysis: You will make two graphs in order to analyze your data. You will graph the data in two different line graphs. Put the amount of acid added along the X axis of one graph, and the amount of base added along the X axis of the second graph, as shown below. On the Y axis, you will graph the pH. You don’t want to have your pH range all the way from 0 to 14—just plot slightly above and below your lowest and highest pH measurements (you will have different pH ranges for the HCl graph and the NaOH graph). Each graph should have 2 lines indicating the change in pH for the 2 different solutions being tested (water, and either

NaH2PO4 buffer or Tris buffer). Unlike the graphs below (these are just present as an example), each of your graphs should take up at least a half a page in your lab notebook. Label each line on the graph with the name of the solution that was being tested.

pH pH

0 20 40 60 80 100 0 20 40 60 80 100

µl HCl µl NaOH

Activity 4d DNA Isolation from Strawberries

Purpose In this activity you will isolate and observe DNA from strawberries. In the process, you will learn how different solutions can act to accomplish different functions in working with biological molecules.

Background This is a simple, effective protocol for spooling DNA. Strawberries are an excellent source for extracting DNA because they are easy to pulverize and contain enzymes called pectinases and cellulases that help to break down cell walls. And most important, strawberries have eight copies of each chromosome (they are octoploid), so there is a lot of DNA to isolate.

Procedure 1. Each group should obtain the following materials: • 3 strawberries • 10 ml DNA Extraction Buffer (soapy salty water) • About 20 ml ice cold 91% or 100% ethyl alcohol (this will be in the freezer until it is needed in order to keep the temperature cold) • 1 Ziploc bag • 1 glass test tube • 1 funnel lined with a moistened paper towel • 1 glass spooling pipette 2. Remove the green sepals from the strawberries. 3. Place strawberries into a Ziploc bag and seal shut. 4. Squish for a few minutes to completely squash the fruit. 5. Add 10 ml DNA Extraction Buffer and squish for a few more minutes. Try not to make a lot of soap bubbles. 6. Filter through a paper towel set in a funnel, and collect the liquid in a clear tube. Do not squeeze the paper towel. Collect about 3-4 ml liquid. Use a 5 or 10 mL pipet to measure the volume of strawberry extract you have collected. 7. Add 2 volumes (2 X the volume of strawberry extract that you’ve collected) ice cold ethyl alcohol to the strawberry liquid in the tube. Pour the alcohol carefully down the side of the tube so that it forms a separate layer on top of the strawberry liquid. 8. Watch for about a minute. What do you see? You should see a white fluffy cloud at the interface between the two liquids. That’s DNA! 9. Spin the glass spooling pipette in the tangle of DNA, and pull out the DNA! The fibers are millions of DNA strands. 10. Make sure you understand the purpose of each ingredient in the procedure (see next page for a detailed description).

(continued on next page)

44 The purpose of each ingredient in the procedure is as follows:

Shampoo or dish soap helps to dissolve the cell membrane, which is a lipid bilayer, releasing the DNA.

Sodium chloride helps to remove proteins that are bound to the DNA and neutralizes the charge repulsion that occurs between DNA strands. It also helps to keep the proteins dissolved in the aqueous layer so they don’t precipitate in the alcohol along with the DNA.

Ethanol or isopropyl alcohol pulls out water molecules in the DNA and causes the DNA to precipitate. When DNA comes out of solution it tends to clump together, which makes it visible. The long strands of DNA will wrap around the glass spooling pipette when it is swirled at the interface between the two layers.

45 Lab 4 Homework Name: ______

1. Which gives a more sensitive result in the measurement of pH: pH indicator paper or a pH meter?

2. Ammonia has a pH of 11.5. Seawater has a pH of 8.5. a) Which liquid is more basic?

b) How many times more basic?

3. When preparing a buffer, why is water added in two stages? Why is water not added to the buffering salt up to the desired final volume, all in one step?

4. Looking at the graphs you made in Activity 4c: a) How can you tell (visually) that a solution being tested contains a buffer?

b) Which buffer had better efficacy (resisted changes in pH more effectively), the 0.1 M

NaH2PO4 pH 4.5 buffer, or the Tris pH 8 buffer?