Laboratory Exercise #2: Making solutions for the lab

Required materials

Glucose

NaCl

CaCl2

Tris

EDTA

Glacial Acetic Acid

Electronic balances

Weight boats

Spatulas

Pipet aids

10 mL serological pipettes

250 mL Glass beakers

500 mL, 250 mL, 100 mL & 50 mL Graduated cylinders

250 mL Glass flasks

Reagent bottles – 100 mL and 1 L

Magnetic stirrers and stir bars

Laboratory Tape

Permanent markers

Introduction

Every biotech and molecular biology labs requires the use of specific solutions. There are two kinds of solutions that are used in bio labs – a stock solution and a working solution. A stock solution is usually a concentrated solution that is stored in the lab and diluted a specific amount to produce a working solution. A working solution is the solution used in the laboratory procedure. Many stock solutions need to be made from powder or from concentrated solutions purchased from commercial sources. To make stock and working solutions, you must have an understanding of molarity, concentration, ratios and dilution.

In this lab, you will make some very basic stock solutions used in most biology labs. You will do this by measuring out specific amounts of solids and combining them with specific volumes of water to make a specific molarity. You will also make working solutions by diluting stock solutions. Finally, you will also make working solutions using percent concentrations and weight to volume (w/v) ratios.

Laboratory Exercise #2.1: Preparing a stock solution (50X TAE)

A scientist makes solutions by dissolving solutes into solvents. A solute is the dissolved matter in solution, while a solvent is a liquid that can dissolve a solute. Of the many types of solutions found in a lab, most are aqueous and use water as the solvent. Distilled water or “ultrapure water” that has been passed through a lab water purification system, such as a Millipore lab water purification system, should be used for making solutions. The sinks here at WLAC have spouts with white handles. These spouts deliver distilled water that is suitable for making solutions. Note: de-ionized water is not suitable for most lab applications.

Scientists follow standard methods to make a solution so that the steps can be replicated by others. These steps should be written in your notebook. The steps essentially become a recipe for all others in your lab.

So making a solution from a solid compound is a lot like baking from a recipe. Baking – not cooking, because you have to follow the recipe EXACTLY.

In this exercise, you will be making 100 mL of a solution called 50X TAE (Tris, Acetic acid, EDTA). 50X TAE will be used throughout this course to make DNA gels and their running buffer.

To make a solution properly, you do not add the solids to the final solution volume. This is because there will be a volume increase as the solids dissolve in the solvent. Some solutions may also need to have their pH adjusted. The general rule of thumb is to add the solid to 80% of the final solution volume, let the solids dissolve (adjust the pH if required) and then determine the volume of the resulting solution. Once you have done this, you add water up to the final volume.

Protocol – wear gloves

  1. To 80 mL of H2O in a 250 mL beaker, add the following:
  2. 24.2 g of Tris
  3. 3.72g of EDTA
  4. Add a magnetic stir bar and place the beaker on a magnetic stirrer. Stir gently until the solids have dissolved. If a magnetic stirrer is not available, you can add the solids to 80 mL water in a 250 mL glass flask and gently swirl to dissolve. Alternatively, you can add the solids to 80 mL of water in a glass reagent bottle, close the bottle and shake to dissolve.
  5. Once dissolved, take the solution to the fume hood. In the fume hood, add 5.7 mL of Glacial Acetic Acid using a pipet aid and a 10 mL serological pipet.
  6. Back at your bench, pour the solution into a 100 mL graduated cylinder and note the volume. Add water until you reach 100 mL.
  7. Carefully pour the prepared 50X TAE into a glass reagent bottle, close the bottle and label it using laboratory tape and a permanent marker. On this bottle, be sure to write the label “50X TAE”, the date of preparation and the initials of who prepared it. Give the bottle to your professor. You will use it to make your DNA gels in this course.

Laboratory Exercise #2.2: Preparing a molar solution (Molarity)

By definition, a mole (mol) of a substance has the same number of molecules as 12 g of carbon and a value of 6.02 x 1023 molecules – a number also known as Avogadro’s number. The atomic mass of an element is denoted in grams and is equal to one mole of the atoms of that element. By adding the atomic masses of the atoms in a compound, the molar mass (or molecular weight) can be determined.

For example, to determine the mass of 1 mole of NaCl (i.e. the molar mass), all you do is add the atomic mass of Na (22.99) and the atomic mass of Cl (35.45). Therefore, the molar mass of NaCl is 58.44 g/mol. This means that one mole of NaCl weighs 58.44 g. To determine the mass of 1 mole of CaCl2, you would add the atomic mass of Ca (40.08) to the atomic mass of two Cl (35.45 x 2 = 70.9). The molar mass of CaCl2 is 110.98 g/mol.

The molecular weight of solid compounds can be found as part of the label.

To make a 1 molar solution of NaCl (denoted as 1 M), you would take one mole of NaCl and add it to 1 liter of H20. This would result in a solution with the molarity of 1 M. But what balance is capable of weighing in moles? None. However, you know that 58.44 g of NaCl represents the weight of 1 mole of this compound. Therefore a 1M NaCl is made by taking 58.44 g of NaCl and dissolving it in 1 liter of H2O. It is important to realize that mole and M are NOT the same thing. A mole refers to the specific number of molecules, while molar is a measure of concentration. The unit of a mole is the gram. The unit of molar is M.

Most of the time, it is unreasonable to make a liter of a solution. Many lab solutions are only 100 mL or 10 mL. To determine the weight of CaCl2 to add to these smaller volumes involves using the proportional method.

For example, if 110.98 g of CaCl2 in 1 liter is a 1 M solution. Then 11.098 or 11.1 g of CaCl2 in 100 mL is also a 1 M solution.

110.98 g CaCl2 =110.98 g CaCl2= 11.1 g CaCl2

1 L 1000 mL 100 mL

But what if you are asked to make 100 mL of a 0.35 M CaCl2 solution? This requires a method known as the unit cancellation method. In this method, the desired molarity is multiplied by the volume unit conversion and then multiplied by the molar mass and then multiplied by the desired volume.

For example, how would you make 100 mL of a 0.35 M CaCl2 solution?

0.35 mole x 1L x 110.98g x 100 mL = 3.88 g of CaCl2would be added to 100 mL of water.

1L 1000 mL 1 mole

NOTE: If the desired volume is the liter, then the volume unit conversion of 1L/1000mL is not needed.

Many lab solutions are not just made of only one compound. For example, one popular solution is called TE Buffer. This solution is made of salts called Tris and EDTA. The standard TE recipe is 10 mMTris and 1 mM EDTA. To make this buffer means making a solution that contains not only 10 mMTris but also 1 mM EDTA in the same volume.

In this exercise, you (and your lab partner) will choose one of the following molar solutions and prepare it.Before you make any solution, determine in your notebook how much solid you will weigh out. Once you have verified your calculations with your professor, then you may make the solution.

  1. 100 mL of 5 M NaCl
  2. 100 mL of 1 M Tris
  3. 100 mL of 0.5 M CaCl2

Protocol – wear gloves

  1. To 80 mL of H2O in a 250 mL beaker, add the correct amount of solid.
  2. Add a magnetic stir bar and place the beaker on a magnetic stirrer. Stir gently until the solids have dissolved. If a magnetic stirrer is not available, you can add the solids to 80 mL water in a 250 mL glass flask and gently swirl to dissolve. Alternatively, you can add the solids to 80 mL of water in a glass reagent bottle, close the bottle and shake to dissolve.
  3. Once dissolved, pour the solution into a 100 mL graduated cylinder and note the volume. Add water until you reach 100 mL.
  4. Carefully pour the prepared solution into a glass reagent bottle, close the bottle and label it using laboratory tape and a permanent marker.

Laboratory Exercise #2.3: Preparing a percent solution

Percent solutions are based on a percentage of the solute in solution using the concepts of 100 parts. Therefore, a 1% solution has 1 part of solute in 100 parts of solvent. Percent solutions are expressed either as “mass or weight per volume” (m/v or w/v) if it is made from a solid or “volume per volume” (v/v) if it is made from a liquid. The mass or weight per volume measurement describes the amount of solid dissolved in a volume of solvent (usually 100 mL). A volume per volume measurement describes the dilution of a liquid solute.

For example, a 5% NaCl (w/v) solution would be made by dissolving 5 g of NaCl in a final volume of 100 mL. To be specific, you would take 5 g of NaCl and dissolve it in 80 mL of H20 and then bring the final volume up to 100 mL. It is possible that you will be asked to make a percent solution in a volume that is not 100 mL. The calculation is shown below.

5.0 g = X g

100 mL 37.0 mL

X = 5.0 g x 37.0 mL = 1.9 g

100 mL

As another example, a 5% (v/v) 2-propanol solution would be made by taking 5.0 mL 2-propanol and adding it to 95.0 mL water. If you wanted to make 37 mL of a 5% 2-propanol solution, you would make it the same way you made the NaCl solution above, except grams would be replaced by mL.

In this exercise, your lab team will choose one of the following percent solutions and prepare it.Before you make any solution, work out your weights or volumes in your notebook. Once you have verified your calculations with your professor, then you may make the solution.

  1. 100 mL of 10% SDS
  2. 100 mL of 10% glucose
  3. 100 mL of 0.9% NaCl

By now, you should be able to make these solutions without a protocol being provided to you. Instead, you will write out your own protocol in your notebook and then prepare the chosen solution.

Laboratory Exercise #2.4: Diluting a stock solution

Many of the solutions you will work with in this course will be provided as a concentrated stock solution. A great example is the 50X TAE solution that you just made. This 50X TAE solution is 50-times more concentrated then the concentration that you will use. A 10X solution is 10-times more concentrated than its working concentration. The equation C1V1 = C2V2calculates the quantity of concentrated solution to make the 1X final solution.

For example, a 10X solution of phospho-buffered solution/PBS needs to be diluted (10-fold) to a 1X concentration in order to be used. If you are asked to make 100 mL of this 1X PBS solution, what do you do?

C1V1 = C2V2, where C is the concentration of the stock solution

10X PBS x V1 = 1X PBS x 1.0 L (1000 mL)

V1 = 1X PBS x 1.0 L

10X PBS

V1 = 0.04 L or 40 mL of PBS + 960 mL of water (1 L total)

Note: if V2 is measured in liters, than V1 is measured in liters.

This formula is very versatile and can be used to calculate how to dilute any solution from a more concentrated one. The C may be a concentrated solution written as a “X” or a concentrated solution written as a %. The V doesn’t necessary have to be in liters. This formula can be used to prepare small volume dilutions (i.e. in uL). As long as you realize that V1 and V2 must be the same units.

A similar formula can be used for diluting molar solutions.

M1V1 = M2V2, where M is the molarity of the stock solution

In this exercise, your lab team will choose one of the following stock solutions and dilute it.Before you make any solution, work out your volumes in your notebook. Once you have verified your calculations with your professor, then you may make the solution.

  1. 250 mL of 1% glucose from a 10% glucose stock
  2. 1 L of 1X TAE from a 50X TAE stock
  3. 100 mL of 70% ethanol from a 95% ethanol stock
  4. 100 mL of 50 mM CaCl2 from 0.5M CaCl2 stock
  5. 100 mL of 10mM Tris from a 1M stock

Write out your own protocol in your notebook and then prepare the chosen solution. Once you have prepared these solutions, label them properly and give them to your professor for future use.

Laboratory Exercise #2.5: Adjusting a solution’s pH

Many solutions need to have their pH adjusted before use. To do this requires the use of a pH meter. Refer to the chapter on lab equipment in order to familiarize yourself with its calibration and use.

In this exercise, you will prepare one of two solutions that will need pH adjustment. Before you do this, you need to calibrate the pH meter and remember how to pH a solution.

Calibration Protocol

The pH meter in this lab is made by Oakton. Like all pH meters it needs to be calibrated before being used. You will perform a two-point calibration by choosing two pH buffers that flank the desired pH of the solution you are making.

  1. Remove the pH meter from its 3M KCl storage solution and rinse it with deionized tap water.
  2. Gently blot the end of the electrode with a paper towel. Do NOT wipe the electrode dry as it will cause static and create calibration and measurement problems.
  3. Make sure the pH meter is in measurement mode.
  4. Choose the pH buffer 7 and pour some into a clean container.
  5. Immerse the electrode (and temperature probe) in the buffer. Make sure the glass bulb of the electrode is completely immersed in the sample. Press the CAL/MEAS to enter calibration mode. The upper display will read “CAL CON”.
  6. When the READY indicator displays in the left hand corner, press the CON button. The pH 7 calibration point is now stored in the meter.

Note: The display will change so that the larger number corresponds to the calibration buffer value, at or around pH 7.00 and the smaller display changes to the next buffer value (pH 4).

  1. If you are satisfied with the calibration, proceed to the pH 4 calibration (the meter assumes the next point is pH 4). If the display changes more than a digit (0.01 pH) from the pH 7 calibration value, repeat the pH 7 calibration.
  2. Calibrate using the pH 4 buffer. The meter will display this value on the smaller display.

Note: If you wish to calibrate using the pH 10 buffer rather than the pH 4 buffer, use the up or down arrow keys to select pH 10 in the smaller display and continue with the next few steps using the pH 10 buffer rather than the pH 4 calibration buffer.

  1. Rinse the electrode with deionized water and immerse it in the pH 4 buffer (or pH 10).
  2. When the READY indicator displays in the left hand corner, press the CON button. This will store the pH of the calibration buffer in the meter.

Note: As with the pH 7 calibration buffer, the larger number will correspond to the pH of the calibration buffer and the smaller display will change to the next buffer value (pH 10).

  1. Press the CAL/MEAS button to exit the calibration mode and begin measuring.

Use the pH meter to determine the pH of a sample of diet soda

  1. Make sure the meter is in MEAS mode. If not, switch it to this mode by pressing the CAL/MEAS button until “MEAS” appears in the upper part of the display.
  2. Immerse the rinsed electrode into the diet soda sample. Make sure the glass bulb of the electrode is completely immersed in the sample.
  3. Stir the electrode gently in the sample.
  4. Take a reading. When the readings stabilize, a READY indicator displays. The READY mode shows the readings are stable within a range of ±0.01 pH.
  5. When you are done taking your reading, press the HOLD button to freeze the displayed reading value.
  6. Rinse the electrode with water, gently blot off the excess liquid and return to electrode to its 3M KCL solution.

In this exercise, your lab team will choose one of the following stock solutions and adjust its pH.Before you make any solution, work out your volumes in your notebook. Once you have verified your calculations with your professor, then you may make the solution.

  1. 100 mL of 10 mMTris-Cl, pH 7.6 (adjust the pH with 12N HCl)
  2. 100 mL of 500 mM EDTA, pH 8.0 (adjust the pH with NaOH pellets)
  3. 100 mL of PBS (adjust the pH with 12N HCl)

Protocol – wear gloves

  1. 10mM Tris-Cl, pH 7.6:
  1. Using your 1M Tris stock, take 10 mL of 1M Tris and transfer it into a glass beaker.
  2. Add 80 mL of water and mix for a volume of 90 mL.
  3. Adjust this solution’s pH to 7.6 using concentrated HCl.
  4. Once you reach the pH of 7.6, transfer the resulting 10 mMTris-Cl to a graduated cylinder and bring the volume to 100 mL with water.
  5. Transfer the adjusted 10 mMTris-Cl to a reagent bottle and label it with your team’s initials and the date.
  1. 500 mM EDTA, pH 8.0:
  1. To 80 mL of water in a glass beaker, add 18.16 g of Na2EDTA2H2O (EDTA).
  2. Put a magnetic stirrer in the beaker and place on a stir plate or gently swirl by hand to dissolve as much EDTA as possible. You will notice that not all the EDTA will dissolve in the water. This is because the pH of EDTA must be adjusted for complete dissolution.
  3. Adjust the pH of the EDTA solution using NaOH pellets (about 2 g will be needed). Add 1.5 g of NaOH pellets to the solution and mix until the pellets have dissolved. Measure the pH.
  4. Add the remaining pellets one at a time, checking the pH continuously.

Note: Be sure to completely dissolve each pellet and allow the pH to stablize before adding another one.

  1. Once pH 8.0 is reached, transfer the EDTA solution to a graduated cylinder and bring the volume to 100 mL with water.
  2. Transfer the adjusted 500 mM EDTA to a reagent bottle and label it with your team’s initials and the date.
  1. Phospho-buffered saline/PBS (pH 7.6):
  1. To 80 mL of water in a glass beaker add the following:
  2. 8 g NaCl
  3. 0.2 g KCl
  4. 1.44 g Na2HPO4
  5. 0.24 g KH2PO4
  6. Adjust the pH to 7.6 with concentrated HCl.
  7. Once you reach the pH of 7.6, transfer the resulting PBS to a graduated cylinder and bring the volume to 100 mL with water.

Transfer the adjusted PBS to a reagent bottle and label it with your team’s initials and the date.