User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/01/30

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Preparing Broth and Agar

Objective

During this lab session, solutions of LB/Agar and LB Broth were prepared. A solution of Ampicilin was prepared to be added to the broth and agar.

Procedure

1. The correct concentration for dissolving LB Broth Media into water is 25g per liter of water. The correct concentration for dissolving granulated Agar into water is 20g per liter of water.

- For the LB/Agar solution. 2.51g of LB Broth Media and 2.00g Difco (TM) granulated Agar were dissolved in 100mL of water - For the LB Broth solution, 1.25g of LB Broth Media were dissolved into 50mL of water

The solution are placed in the autoclave at 121 degrees C for an hour.

2. In order to prepare the ampicilin solution, 100mg of Ampicilin Sodium Salt were dissolved into 1mL of sterilized water. This solution was prepared in a sterilized eppendorf tube using sterile equipment.

3. Once the solutions cool, 100uL of the ampicilin solution is added to the LB/Agar solution

4. The LB/Agar solution is now plated. approximately 25mL of the solution is poured into the sterilized plates.

Cell Transformation

Before Starting

    • Equilibrate a water bath at 42 degrees C
    • Warm the vial of SOC medium to room temperature
    • Warm the plates at 37 degrees C in incubator for 30 minutes

Procedure

  1. Thaw on ice, one 50 uL vial of One Shot(R) cells for each ligation/transformation
  2. Pipet 1 to 5uL of each ligation reaction directly into the competent cells and mix by taping gently. Do not mix by pipetting up and down. Store the remaining ligation reaction at -20 degrees C
    1. Two vials were prepared
      • One with 1.66uL of DNA
      • One with 3.33uL of DNA
  1. Incubate the vials on ice for 30 minutes
  2. Incubate for exactly 30 seconds in the 42 degree C water bath. Do not mix or shake.
  3. Remove vial from water bath and place on ice.
  4. Add 250uL of pre-warmed SOC medium to each vial (SOC iix a rich medium sterile technique must be practiced to avoid contamination.)
  5. Place the vial in a micro centrifuge rach on its side and secure with tape to avoid losing vial. Shake the vial at 37 degrees C for exactly 1 hour at 225rpm in a shaking incubator.
  6. Spread 20 to 200uL from each transformation vial on separate, labeled LB agar plates. WE recomment you plate two different volumes. NB: You may have to dilute cells 1:10 to obtain well-spaced colonies.
  7. Store the remaining transformation reaction at +4 degrees C and plate out the next day if desired.
  8. Invert plates and incubator at 37 degrees C overnight
  9. Select colonies and analyze plasmid isolation, PCR, or sequencing