12: Advance Solution Preparation

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Chapter Twelve

Advance Solution Preparation

Let's discuss the importance of precision and accuracy in scientific research, particularly when it comes to preparing solutions for experiments and assays. When we talk about advanced solution preparation, we're referring to careful attention to detail, following standard operating procedures (SOPs) to a tee, and using specialized equipment to ensure that our results are consistent and trustworthy. Today, we're going to explore the step-by-step procedures (SOPs) for preparing some common laboratory solutions that you'll encounter in your scientific journey. These include media for growing microorganisms, buffers with carefully adjusted pH levels, and gels used in a technique called electrophoresis.

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LAB ACTIVITY - PART 1 - Preparation with Autoclave

Autoclaving:

Let's talk about autoclaving, which is a fundamental step in preparing laboratory solutions. Autoclaving is crucial because it sterilizes our equipment, media, and solutions, getting rid of any pesky microbial contaminants that could mess up our experiments. How does it work? Well, we use an autoclave, which is like a pressure cooker or a steam machine. It heats water to produce high-pressure steam, and we subject our solutions to this steam at temperatures of about 121°C for a set amount of time, usually between 15 to 30 minutes. This high temperature and pressure combo effectively kills bacteria, fungi, and other microorganisms, ensuring that our lab materials are sterile and ready for use in our experiments.

Alright, let's walk through the process of preparing LB broth and agar plates for growing bacteria, and then how we autoclave them to ensure they're sterile and ready for our experiments.

The objective is to prepare 50mL of Luria-Bertani (LB) broth and 30mL of Luria-Bertani (LB) agar plates using autoclave sterilization.

Materials:

• 125mL Erlenmeyer Flask (Carolina, #721149A)
• Tryptone (Carolina, #216741)
• Yeast Extract (Carolina, #216746)
• NaCl (Flinn Scientific, #S0064)
• Aluminum foil (Carolina, #974844)
• LB Broth Agar Base (Carolina, 216701)
• Petri dish (Carolina, #741251)
• Autoclave

50mL of Luria-Bertani (LB) Broth Preparation:

• First, we need to gather our materials, including a 125mL Erlenmeyer flask.
  • Each group gathers one 125mL Erlenmeyer Flask.
  • Ensure the availability of tryptone, yeast extract, NaCl, and aluminum foil.
• We'll add the ingredients for LB broth: 1% tryptone, 0.5% yeast extract, and 1% NaCl. These provide the nutrients bacteria need to grow.
  • Measure Ingredients:
    • Add 0.5g of tryptone (1% concentration) into the Erlenmeyer Flask.
    • Add 0.25g of yeast extract (0.5% concentration) into the same flask.
    • Add 0.5g of NaCl (1% concentration) into the flask.
• Gently swirl the flask to ensure a thorough mixing of the ingredients.
  • The solution won’t dissolve completely until after you autoclave.
• To prevent any contamination, we'll cover the flask with aluminum foil.
• Now, it's time to sterilize our LB broth. We'll place the flask in the autoclave and set it to run at 121°C for 25 minutes.
  • Cap the flask tightly with aluminum foil to prevent contamination during autoclaving.
  • Place the capped flask in the autoclave, and autoclave for 25 minutes at 120°C.
• After autoclaving, we need to let the LB broth cool down to room temperature before we can use it to grow bacteria.

30mL Luria-Bertani (LB) Agar Plate Preparation:

• Similarly, we'll prepare our LB agar solution in another 125mL Erlenmeyer flask.
  • Each group gathers one 125mL Erlenmeyer Flask.
  • Ensure the availability of LB Broth Agar Base and aluminum foil.
• This time, we'll add 3% Luria Broth Agar Base to the flask. Agar makes the solution solid, providing a surface for bacteria to grow on.
  • Add 0.9g of LB Broth Agar Base (3% concentration) into the Erlenmeyer Flask.
  • Add 30mL of distilled water to the flask.
  • Swirl the flask gently to dissolve the agar base.
    • The solution won’t dissolve completely until after you autoclave.
• Just like before, we'll cover the flask with aluminum foil to keep it clean.
• The flask goes into the autoclave, again at 121°C for 25 minutes, to make sure it's sterile.
  • Cap the flask tightly with aluminum foil to prevent contamination during autoclaving.
  • Place the capped flask in the autoclave, and autoclave for 25 minutes at 120°C.
• After autoclaving, we need to cool the agar solution to around 42°C. This temperature is important because it's hot enough to keep the agar liquid, but not so hot that it damages any heat-sensitive components and molecules.
• Once cooled to touch, we pour the agar solution into sterile petri dishes, where it will solidify. These plates are now ready for use in our bacterial culture experiments.
  • Carefully pour the cooled agar solution into petri dishes.
  • Leave the plates undisturbed until the agar solidifies.

So, there you have it! With these steps, we can prepare LB broth and agar plates, autoclave them to ensure they're free of contaminants, and create the perfect environment for growing bacteria in our experiments.

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LAB ACTIVITY - PART 2 - Preparation with pH Adjustment

Next, let’s dive into pH adjustment, an essential skill for any scientist working in the lab. Whether you're conducting experiments in biochemistry or microbiology, understanding how to fine-tune pH levels is crucial for maintaining the right conditions for reactions to occur. Why do we care about pH adjustment? Well, imagine trying to bake a cake without measuring the ingredients properly. Just like how the right amount of sugar and flour is essential for a perfect cake, the correct pH level is crucial for many experiments to work effectively. So, what exactly is pH adjustment? It's the process of adding either acids or bases to a solution to reach a specific pH level. Once we've added the acids or bases, we need to mix everything up thoroughly and then double-check our work using a pH meter or pH indicator.

The objective is to prepare solutions with specific pH levels using Tris buffer and Sodium Acetate/Acetic acid buffer solutions.

Materials:

• Tris (Flinn Scientific, #FB2393)
• 0.1M Sodium Acetate solution
• 0.1M Acetic acid solution
• Distilled water
• Serological pipettes
• Micropipettes
• Beakers
• pH meter
• Stirring rod or magnetic stirrer

Procedure: Preparation of Tris Buffers

Let's break it down with an example: Your goal is to create 0.5M Tris buffer solution at pH 4, pH 7, and pH 10. Tris buffers are super versatile and can maintain a stable pH across different temperatures, making them perfect for many biochemical and molecular biology experiments. Here's a step-by-step guide, or Standard Operating Procedure (SOP), for preparing Tris buffers:

• Step 1: Gather your materials and prepare a 20mL of 0.5M Tris base solution.
  • Using a digital balance, weigh out the appropriate amount of Tris into a clean beaker
    • 1.21g of Tris base
  • Add distilled water to the Tris base, bringing the total volume to 16mL (80% of the total).
  • Stir the solution gently until the Tris base is completely dissolved.
• Step 2: Using a pH meter, measure the pH of each Tris solution and adjust the pH using either hydrochloric acid (HCl) to lower the pH or sodium hydroxide (NaOH) to raise the pH. Record the volume of acid or base added to adjust the pH.
• Step 3: Once you've hit your target pH, adjust the volume of the solution to 20mL with distilled water. Give it a good mix, then label your bottles clearly with the pH and Tris concentration.

Procedure: Preparation of Sodium Acetate/Acetic Acid Buffers

Let's do another example, but this time the buffer has two concentrations from different chemicals: Your goal is to create 0.1M sodium acetate/0.1M acetic acid solution at pH 5.5 and 7.5. Here's a step-by-step guide, or Standard Operating Procedure (SOP), for preparing a buffer with two separate concentrations:

• Step 1: Gather your materials and prepare a 10mL of 0.2M Sodium Acetate solution.
• Step 2: Gather your materials and prepare a 10mL of 0.2M Acetic acid solution.
• Step 3: Mix the two solutions together to obtain 20mL of volume.
• Step 4: Stir the solution gently and use a pH meter to adjust the pH using either HCl or NaOH until the desired pH of 5.5 or 7.5 is achieved. Record the volume of acid or base added to adjust the pH.
• Step 5: Once you've hit your target pH, adjust the volume of the solution to 20mL with distilled water. Give it a good mix, then label your bottles clearly with the pH and Tris concentration.

And there you have it! That's the basics of pH adjustment in the lab. Remember, practice makes perfect, so don't be afraid to get your hands dirty and experiment with different pH levels. Who knows, you might just discover the next big breakthrough in science!

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LAB ACTIVITY - PART 3 - Preparation of Stock and Gel

Finally, let's dive into gel preparation, a crucial step in the realm of electrophoresis. Electrophoresis helps us separate DNA, RNA, or proteins based on their size and charge. Today, we're focusing on agarose gels.

Imagine you're in the lab, ready to separate DNA fragments using agarose gel electrophoresis. You probably want to make sure there’s a buffer that can support the high-voltage current. TBE Buffer ensures a stable pH and provides the necessary ions for the electric performance during electrophoresis. Here's a step-by-step guide, or Standard Operating Procedure (SOP), for preparing the agarose gel and the required buffer:

The objective is to prepare solutions of calcium chloride, 1X TBE buffer, and agarose gels in 1X TBE buffer.

Materials:

• Calcium Chloride (Carolina, 851840)
• Tris base (Flinn Scientific, #FB2393)
• Boric acid (Flinn Scientific, #B0081)
• EDTA (Gold Biotechnology, #E-210)
• Agarose powder
• Distilled water
• Serological pipette or Micropipette
• Beakers
• Magnetic stirrer or stirring rod
• pH meter
• Microwave or hot plate
• Gel electrophoresis chamber

Procedure: Preparation of 1X TBE Buffer

*Step 1: Preparation of 30mL of 1X TBE Buffer, pH 8

• Weigh to obtain 0.1M Tris, 0.1M boric acid, and 0.002M EDTA.
  • *Uh Oh! It’s extremely difficult to accurately weigh out the 0.002M EDTA. Thus, it is recommended that we start with a higher concentration of TBE buffer, and then dilute it later.

Step 1: Preparation of 30mL of 10X TBE Buffer, pH 8

• Let’s start again: Weigh to obtain 1M Tris, 1M boric acid, and 0.02M EDTA.
• Dilute the mixture to a final volume of 30mL with distilled water.
• Stir the solution thoroughly until all components are dissolved.
• Check the pH of the solution using a pH meter and adjust if necessary using concentrated HCl or NaOH solutions.
• Don't forget to label your bottle clearly as 10X TBE buffer, pH 8.

Step 2: Dilute 10X to 1X TBE Buffer, pH 8

• Dilute the solution 10-fold, by transferring 3mL into 27mL distilled water to obtain 1X TBE buffer.
  • Apply C1V1=C2V2 to create 30mL of 1X TBE buffer, pH 8.

Step 3: Preparation of 0.8% Agarose Gels

• Measure out 30mL of the 1X TBE buffer prepared from the previous steps.
• Add 0.8% of agarose to the TBE buffer solution.
  • Measure 0.24g of agarose powder for each 30mL 1X TBE buffer into a clean container.
• Heat the mixture in a microwave (40 seconds interval) or hotplate until the agarose is completely dissolved, stirring occasionally.
• Let the solution cool to around 42°C before pouring it into a gel tray.
• Insert a comb into the gel to create wells – the special spots for sample loading.
  • Pour the solution into a gel tray and insert a comb to create wells for sample loading.
• Allow the gel to solidify at room temperature for roughly 30 minutes.

The agarose solution, now solidified, forms a gel matrix with wells, ready for molecular biology experiments. Once the gel is solidified, we place the gel into the electrophoresis chamber filled with 1X TBE buffer, creating a conducive environment. Then, our DNA samples can be loaded into the wells for their separation.

Other: Preparation of 0.2M Calcium Chloride Solution

• Create a stock solution of Calcium Chloride
  • Combine 1.11g of calcium chloride with 10mL of distilled water in a clean beaker
  • Stir the solution until the calcium chloride is completely dissolved.
• Preparing 1mL of 0.1M Calcium Chloride:
  • Dilute and aliquot from the calcium chloride stock solution to obtain 1mL of 0.2M Calcium chloride solution.

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LAB ACTIVITY - PART 4: Practicum 2 (60 points total)

    This evaluation will assess your proficiency in various laboratory techniques. You will be split into TWO teams. Work together to complete all projects efficiently and accurately. Work together within your team to plan out each task. Begin assigning tasks for every individual! Please follow the Standard Operating Procedures (SOP) from the previous class carefully. Your team will create one of each buffer from the list below. Ensure all submitted buffers are labeled, otherwise, the buffer will be 0 points

Objective:

• Team Buffer-Making Challenge: This practicum tests your hands-on lab skills and your ability to work as a team under time pressure.
• You will work in teams to create 10 different buffers/solutions using only the provided SOPs—no pH meters allowed! Your solutions must be labeled clearly to receive credit.