Sign InSign Up

Preparing glycerol stocks of K. rhaeticus

Updated 6/6/2019 09:34 PM
Step-by-step
by SoundBio iGEM (created by Yoshi Goto)

Introduction

Based on Imperial College iGEM's paper https://www.pnas.org/content/113/24/E3431

Materials

  • K. rhaeticus
    • 50mL Tube
      • HS Media
        • Cellulase
          • Glycerol
            • 2mL Cryostock tube

              Procedure

              hell yeah glycerol stocks

              1. Before you start: make sure glucose has been added to HS media after the glucose and HS have been autoclaved separately. Add equal volume of 4% glucose to HS without glucose. See this protocol for instructions on how to prepare HS media.
              1. Put 5mL of HS media into a 50mL tube.
              1. Inoculate the HS media with K. rhaeticus.

              If using a previous glycerol stock, tap a p1000 pipette tip to the frozen stock. Eject the tip into the liquid media.

              If using a colony from a plate, gently touch a single colony with a p1000 pipette tip. Eject the tip into the liquid media.

              1. Incubate without shaking at 30 C for 6 days
              1. After 6 days, add cellulase to 0.2% (vol/vol) (10µL for a 5mL culture)
              1. Now incubate with shaking at 30 C at 230 rpm for 1 day.
              1. Now add glycerol to 25% (vol/vol) (so for a 5mL culture, add 1.667mL for a total of 6.667mL)
              1. Make 1.5mL aliquots of the culture in 1.8mL cryostock tubes and store at -80 C. Update the iGEM organism database.

              Comments

              Benchling
              Learn More
              Contact
              • Contact Us
              • 680 Folsom St, 8th Floor
                San Francisco, CA 94107
              Legal

              Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. It doesn't look like your current browser is supported - for more information, click here.