How to Make Acrylamide Bisacrylamide Solution: A Comprehensive Guide

Introduction to Acrylamide Bisacrylamide Solution

Acrylamide and bisacrylamide are commonly used chemicals in the field of biochemistry, particularly in the preparation of polyacrylamide gels for electrophoresis. The mixture of these two chemicals in specific ratios is crucial for creating gels with desired properties, such as porosity and mechanical strength. Understanding how to make acrylamide bisacrylamide solution is essential for researchers and technicians who frequently work with electrophoresis.

Importance of Proper Ratios in Acrylamide Bisacrylamide Solutions

When making acrylamide bisacrylamide solution, the ratio between acrylamide and bisacrylamide is critical. Acrylamide serves as the monomer that polymerizes to form the gel, while bisacrylamide acts as a crosslinker, linking the acrylamide chains together. The concentration of bisacrylamide relative to acrylamide determines the pore size of the gel. For example, a 19:1 acrylamide to bisacrylamide ratio creates a gel with larger pores, suitable for separating large molecules, whereas a 29:1 ratio results in a gel with smaller pores, ideal for smaller molecules.

Materials and Equipment Required

To make acrylamide bisacrylamide solution, you will need the following materials:

1. Acrylamide: Typically, a 30% (w/v) acrylamide solution is used as the starting material.
2. Bisacrylamide: Usually provided as a solid, it needs to be dissolved in water to prepare a stock solution.
3. Deionized Water: Ensures no contaminants interfere with the polymerization process.
4. Buffer Solution: Tris buffer is commonly used to maintain the pH at the desired level during gel formation.
5. Stirring Equipment: A magnetic stirrer and a clean stir bar for thorough mixing.
6. Volumetric Flask or Beaker: For precise measurement and mixing of solutions.
7. pH Meter: To ensure the solution is at the correct pH, typically around 8.8 for polyacrylamide gels.

Step-by-Step Process for Making Acrylamide Bisacrylamide Solution

1. Preparation of Stock Solutions

• Begin by preparing a 30% (w/v) acrylamide stock solution by dissolving the required amount of acrylamide in deionized water. It’s essential to handle acrylamide with care, as it is a neurotoxin. Always wear appropriate PPE.
• Prepare a 1.6% (w/v) bisacrylamide stock solution by dissolving the bisacrylamide in deionized water. This concentration is typical but may vary depending on the specific gel requirements.

2. Mixing the Acrylamide and Bisacrylamide Solutions

• To create the acrylamide bisacrylamide solution, measure the desired volume of acrylamide stock solution based on the total volume needed for your gel.
• Add the appropriate amount of bisacrylamide stock solution to achieve the desired ratio (e.g., 29:1 or 19:1).

3. Adjusting the Solution's pH

• After mixing, add the Tris buffer to the acrylamide bisacrylamide solution to adjust the pH. Use a pH meter to ensure the pH is around 8.8, which is suitable for most gel electrophoresis applications.

4. Final Adjustments and Filtration

• Once the pH is adjusted, make up the volume to the desired level with deionized water. Stir the solution until it is homogenous.
• Filter the solution using a 0.45 µm membrane filter to remove any particulate matter. This step ensures the solution is clear and free of impurities, which could affect gel polymerization.

Storage and Handling of Acrylamide Bisacrylamide Solution

After preparation, store the acrylamide bisacrylamide solution in a dark bottle at 4°C to prevent degradation. Exposure to light or high temperatures can cause premature polymerization, rendering the solution unusable. Label the bottle clearly with the concentration, date of preparation, and any safety warnings.

Conclusion

Knowing how to make acrylamide bisacrylamide solution is fundamental for researchers working with polyacrylamide gels. By following the proper steps—preparing stock solutions, mixing in the correct ratios, adjusting pH, and filtering—you can produce a high-quality solution that meets your experimental needs. Proper storage is also crucial to maintain the stability and effectiveness of the solution. Whether you're preparing gels for protein or nucleic acid separation, mastering the preparation of acrylamide bisacrylamide solution is key to consistent and reliable results in the lab.