Protocol for Making LB Plates for Bacterial Culture (Kanamycin Antibiotic)

Materials: 

• Tryptone: 10 g/L

• Yeast Extract: 5 g/L

• Sodium Chloride (NaCl): 10 g/L

• Distilled Water: (900mL)

• Kanamycin (stock solution, typically 50 mg/mL)

Procedure:

1. Weighing the Components:

2. • Weigh 10 grams of Tryptone.

   • Weigh 5 grams of Yeast Extract.

   • Weigh 10 grams of Sodium Chloride (NaCl).

3. Dissolving the Components:

4. • Add the Tryptone, Yeast Extract, and Sodium Chloride to a beaker or flask.

   • Add approximately 900 mL of distilled water to the beaker.

   • Stir the solution until all components are completely dissolved.

5. Adjusting the Volume:

6. • After the components are dissolved, adjust the final volume of the solution to 1 liter using distilled water.

7. Sterilization:

8. • Transfer the prepared LB medium to suitable containers (e.g., bottles or flasks).

   • Autoclave the medium at 121°C for 15-20 minutes to sterilize.

9. Cooling:

10. • Allow the sterilized LB medium to cool to about 50-55°C (not fully cooled to room temperature but cool enough not to degrade the antibiotic).

11. Adding Kanamycin:

12. • Once the medium has cooled, add kanamycin to a final concentration of 50 µg/mL.

    • For a 1 liter solution, add 1 mL of kanamycin stock solution (50 mg/mL).

    • Mix thoroughly to ensure even distribution of the antibiotic.

13. Storage:

14. • The LB medium with kanamycin can be stored at room temperature or in a refrigerator until needed. If making LB agar plates, pour the medium into petri dishes immediately after adding kanamycin.

Optional:

• If solid LB agar plates are required, add 15 g/L of agar before autoclaving. After autoclaving, cool to about 50-55°C before adding kanamycin and pouring into plates.

This protocol yields 1 liter of LB medium with kanamycin. Adjust the quantities proportionally for different volumes.

Protocol for Making M9 Plates for Bacterial Culture (Kanamycin Antibiotic)

M9 Medium with Kanamycin Preparation Protocol

Materials (per liter):

• Na2HPO4∙7H2O: 12.8 g

• KH2PO4: 3 g

• NaCl: 0.5 g

• NH4Cl: 1 g

• H2O: to 986 mL

• (If making plates, add 15 g of agar)

Add separately before use (per liter):

• 1M MgSO4: 2 mL

• 0.1 M CaCl2: 1 mL

• 50% glycerol: 10 mL

• 1M IPTG: 0.5 mL (optional, only if induction of expression is needed)

• 50 mg/mL kanamycin: 1 mL

Instructions:

1. Dissolve salts:

2. • In a 1 L beaker or flask, add 986 mL of deionized water.

   • Add the following chemicals in sequence, allowing each to dissolve fully before adding the next:

   • • 12.8 g of Na2HPO4∙7H2O

     • 3 g of KH2PO4

     • 0.5 g of NaCl

     • 1 g of NH4Cl

3. Adjust the volume:

4. • If necessary, adjust the volume of the solution to 986 mL with deionized water.

5. Autoclave:

6. • If making liquid M9 medium, autoclave the solution for 35 minutes at 121°C.

   • If making M9 agar plates, add 15 g of agar before autoclaving. Sterilize for 35 minutes at 121°C.

7. Add supplements after cooling:

8. • Once the solution cools to ~55°C (to avoid denaturation of sensitive components), add the following sterile solutions:

   • • 1M MgSO4: 2 mL

     • 0.1 M CaCl2: 1 mL

     • 50% glycerol: 10 mL

     • 1M IPTG: 0.5 mL (optional, only if induction of expression is needed)

     • 50 mg/mL kanamycin: 1 mL

9. Final volume:

10. • Adjust the final volume to 1 L with sterile deionized water if necessary.

11. Storage:

12. • Store the prepared medium at 4°C if not using immediately. If plates were prepared, store them upside down at 4°C.

Your M9 medium with kanamycin is now ready to use for bacterial culture.

Protocol for Making 50 mg/mL Kanamycin Stock Solution (1mL Aliquots)

Materials:

• Kanamycin sulfate powder

• Deionized water (DI water)

• Sterile 1.5 mL microcentrifuge tubes

• 0.22 μm sterile syringe filter or 0.22 μm sterile filter unit

• Pipettes and sterile tips

• Scale for weighing

Steps:

1. Weigh the kanamycin sulfate:

2. • Measure 0.05 g of kanamycin sulfate powder using a clean, calibrated scale.

3. Dissolve in deionized water:

4. • Transfer the kanamycin powder into a sterile container.

   • Add 1 mL of deionized water to the kanamycin powder.

   • Gently mix by pipetting up and down or vortex until the kanamycin is fully dissolved.

5. Sterilize the solution:

6. • Use a 0.22 μm sterile filter to sterilize the kanamycin solution. Draw the solution into a syringe, attach the filter, and expel the solution into a sterile microcentrifuge tube.

7. Aliquot the solution:

8. • Pipette 1 mL of the sterilized 50 mg/mL kanamycin solution into each sterile 1.5 mL microcentrifuge tube.

9. Label the aliquots:

10. • Clearly label each tube with "50 mg/mL Kanamycin," the date, and your initials.

11. Storage:

12. • Store the aliquots at -20°C for long-term use. Aliquots can be thawed as needed.

Note:

• Kanamycin is stable for several months when stored at -20°C. Avoid repeated freeze-thaw cycles by only thawing the aliquot needed.

Protocol for Making 1M IPTG Stock Solution (0.5 mL Aliquots)

Materials:

• IPTG (Isopropyl β-D-1-thiogalactopyranoside) powder

• Deionized water (DI water)

• Sterile 1.5 mL microcentrifuge tubes

• 0.22 μm sterile syringe filter or 0.22 μm sterile filter unit

• Pipettes and sterile tips

• Scale for weighing

Steps:

1. Weigh the IPTG:

2. • Measure 2.38 g of IPTG powder (molecular weight: 238.3 g/mol) using a clean, calibrated scale. This amount will make 10 mL of a 1M solution.

3. Dissolve in deionized water:

4. • Transfer the IPTG powder into a sterile container.

   • Add 10 mL of deionized water.

   • Gently mix by pipetting up and down or using a vortex until the IPTG is fully dissolved.

5. Sterilize the solution:

6. • Use a 0.22 μm sterile filter to sterilize the IPTG solution. Draw the solution into a syringe, attach the filter, and expel the solution into a sterile container.

7. Aliquot the solution:

8. • Pipette 0.5 mL of the sterilized 1M IPTG solution into each sterile 1.5 mL microcentrifuge tube.

9. Label the aliquots:

10. • Clearly label each tube with "1M IPTG," the date, and your initials.

11. Storage:

12. • Store the aliquots at -20°C for long-term use. Aliquots can be thawed as needed.

Note:

• IPTG is stable for several months at -20°C. Avoid repeated freeze-thaw cycles by only thawing the aliquot needed for use.

Protocol for 50% Glycerol Stock Solution

Materials:

• Glycerol (100% or anhydrous glycerol)

• Sterile deionized water (DI water)

• 50 mL conical tube or sterile container

• Pipettes and sterile tips

Steps:

1. Measure the glycerol:

2. • If working with 100% glycerol, it is very viscous, so use a large pipette tip or pour it carefully. Measure 50 mL of glycerol.

3. Add deionized water:

4. • Add 50 mL of sterile deionized water to the glycerol to bring the final volume to 100 mL. This will make a 50% glycerol solution.

5. Mix thoroughly:

6. • Gently mix the glycerol and water until fully combined. This may take a bit of time due to the viscosity of the glycerol.

7. Sterilize (optional but recommended for some applications):

8. • If sterile conditions are required, filter the solution through a 0.22 μm sterile filter to remove any potential contaminants.

9. Aliquot and store:

10. • Aliquot the solution into sterile 1.5 mL or 50 mL tubes as needed. Store the aliquots at room temperature or 4°C.

Note:

• 50% glycerol is commonly used as a cryoprotectant for bacterial and yeast stocks. For long-term storage, cultures are typically mixed with equal parts of the 50% glycerol stock (resulting in a final concentration of 15-20% glycerol) and stored at -80°C.

Protocol for Preparing 1M MgSO₄ (Magnesium Sulfate) Stock Solution

Materials:

• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) powder

• Deionized water (DI water)

• 1 L volumetric flask or beaker

• Scale for weighing

• Stirring rod or magnetic stirrer

• 0.22 μm sterile syringe filter (optional for sterilization)

• Sterile storage container (e.g., 50 mL conical tubes)

Steps:

1. Weigh the magnesium sulfate:

2. • Measure 120.37 g of MgSO₄·7H₂O using a clean, calibrated scale. This will make 1 liter of a 1M solution.

3. Dissolve in deionized water:

4. • Add the MgSO₄·7H₂O to a beaker or volumetric flask containing about 800 mL of deionized water.

   • Stir the solution with a stirring rod or magnetic stirrer until the magnesium sulfate is completely dissolved.

5. Adjust to final volume:

6. • Once the magnesium sulfate is fully dissolved, add deionized water to bring the final volume to 1 liter.

7. Sterilize the solution (optional but recommended):

8. • If the solution needs to be sterile, filter it through a 0.22 μm sterile filter into a sterile container.

9. Aliquot and store:

10. • Transfer the solution into sterile containers or aliquots (e.g., 50 mL conical tubes).

    • Label with "1M MgSO₄," the date, and your initials.

    • Store at room temperature or at 4°C.

Note:

• 1M MgSO₄ solution is commonly used as a supplement in bacterial growth media, such as M9 medium, to provide magnesium ions necessary for various cellular processes.

Protocol for Preparing 0.1M CaCl₂ (Calcium Chloride) Stock Solution

Materials:

• Calcium chloride dihydrate (CaCl₂·2H₂O) powder

• Deionized water (DI water)

• 1 L volumetric flask or beaker

• Scale for weighing

• Stirring rod or magnetic stirrer

• 0.22 μm sterile syringe filter (optional for sterilization)

• Sterile storage container (e.g., 50 mL conical tubes)

Steps:

1. Weigh the calcium chloride:

2. • Measure 14.7 g of CaCl₂·2H₂O using a clean, calibrated scale. This amount will make 1 liter of a 0.1M solution.

3. Dissolve in deionized water:

4. • Add the calcium chloride to a beaker or volumetric flask containing about 800 mL of deionized water.

   • Stir the solution using a stirring rod or magnetic stirrer until the calcium chloride is fully dissolved.

5. Adjust to final volume:

6. • After the calcium chloride has dissolved, add deionized water to bring the final volume to 1 liter.

7. Sterilize the solution (optional but recommended):

8. • If sterility is required, filter the solution through a 0.22 μm sterile filter into a sterile container.

9. Aliquot and store:

10. • Transfer the solution into sterile containers (e.g., 50 mL conical tubes) or smaller aliquots if necessary.

    • Label with "0.1M CaCl₂," the date, and your initials.

    • Store at 4°C.

Note:

• 0.1M CaCl₂ is often used for preparing competent bacterial cells, as calcium ions help cells take up DNA during transformation.