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Latest revision as of 07:25, 13 October 2018

Kanamycin and cortisol agar plate prep · Benchling

Kanamycin and cortisol agar plate prep

Introduction

Addgene.

Materials

  • 37g pre-mixed powder consisting of:
    • 5 g peptone
    • 10.0 g peptone from casein
    • 10.0 g sodium chloride
    • 12.0 g agar-agar
  • 1.0 L Sterile H2O
    • Sterile plates of your desired size (usually 60 mm x 15 mm plates) which can hold 5-10 mL of agar on which you can individually distinguish a maximum of ~200 colonies.
      • Autoclave flasks
        • Sterile pipettes
          • Ice bucket to hold antibiotics
            • Antibiotic at 1000 x concentration dissolved in the appropriate liquid solvent.
              • Kanamycin --> stock concentrationL 50 mg/mL, recommened working concentration (50µg/mL).

            Procedure

            1. Measure 37 g of pre-mixed LB-agar powder per L molten agar you'd like to make. The precise mass you measure out will be based on the number of plates you'd like to pour.
            1. Transfer the LB-agar powder you've measured out into an appropriately sized bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The extra empty volume is necessary to prevent your molten agar from boiling over in the autoclave
            1. Transfer the sterile water to the same bottle and swirl to form a medium/agar colloid
            1. Cover the opening of the bottle with its cap or aluminum foil (but do not make an air-tight seal!) and tape the bottle with autoclave tape. The autoclave tape will darken during the autoclave process if your sample has spent at least 10 min at 121 ℃. Use lab tape to label the bottle with your initials, the date, and the bottle contents. This will clear up any confusion later if your forget your bottle in the autoclave.
            1. Place the gel mix in the autoclave and run on a setting that gets the sample to at least 121 ℃ under 20 psi for at least 30 min. The high pressure will prevent your gel mix from boiling over at high temperature.
            1. While your samples are sterilizing in the autoclave, you should prepare your plate pouring station:
            • - Find an empty section of lab bench with a working flame.
            • - Spray down the bench with a 70% ethanol solution and wipe down with a paper towel.
            • - Count out the appropriate number of plates and stack them on your lab bench.
            • - Label the plates with the date and the medium they will contain including the identity of the antibiotic
            • - Position the flame just to the side of where you’ll be pouring your plates - be sure to leave room for your bottle of molten gel mix, a tube rack containing the appropriate antibiotics, and a section for active pouring.
            1. Prepare your antibiotics. Prior to adding your antibiotic to the molten gel mix, you should create a 1000x stock solution.
            • - For example: If you’ll be preparing plates with a final concentration of 100 ug/mL ampicillin, you should make a stock solution of 100,000 ug/mL (100 mg/mL). Simply measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize.
            1. Prepare a water bath at 60 ℃ with sufficient water to submerge ~75% of the bottle containing your molten gel mixture.
            • - Make sure that kanamycin doesn't breakdown at this temperature.
            1. Retrieve your molten agar mix from the autoclave.
            1. Partially submerge your molten gel-mix in the 60 ℃ water bath.
            • - You should leave the molten gel-mix in the water bath for at least 5 min. Do not let any of the water bath water touch the neck or top of the bottle as this water is not likely to be sterile. Cooled agar should be warm to the touch; as a rule of thumb, if you cannot take the molten agar out of the water bath wearing only lab gloves, it’s not likely cool enough to add antibiotic to. To be certain your agar is at the right temperature, we recommend using a laser thermometer.
            1. Light the flame at the plate pouring station and dilute your antibiotic into your ~60 ℃ molten gel mix using sterile technique.
            1. Swirl the agar bottle to ensure even distribution of the antibiotic throughout the agar.
            1. Open one plate at a time next to the flame and begin pouring. Measure your desired amount of agar with a pipette for the first plate to get a good idea of what that volume looks like in your particular plate.
            1. For the remainder of the plates, pour directly from the bottle.
            • - Be sure to swirl your plates after pouring to remove bubbles and ensure an even distrubtion of agar over the bottom of the plate. Cap each plate after pouring and stack as you pour
            • - If your agar partially solidifies in the bottle while you’re pouring, you should stop pouring and re-make the gel-mix. If you’re making plates without any antibiotic you can alternatively re-liquefy the agar by running it through the autoclave again or by microwaving (if you microwave, beware of over-boiling!).
            1. Leave your plates out on the bench to solidify.
            • - It takes roughly 30 min for our plates to solidify at room temperature, however we leave them out at room temperature overnight to allow them to dry. After overnight drying, we place the plates in a plastic bag with an absorbent material to reduce condensation. The plates are then stored at 4 ℃ until use.
            1. Once your plates have solidified and dried, you should test them to make sure the antibiotic functions properly
            • - Take out two plates.
            • - On the first plate, streak out a strain that you know to be resistant to the antibiotic.
            • - On the second plate, streak out a strain that’s not resistant to the antibiotic.
            • - Incubate both plates overnight at the appropriate growth temperature and check for growth. See our sample data section below for positive and negative test results