Apr 10, 2018

Public workspacePreparing E. coli cryo cultures

DOI
dx.doi.org/10.17504/protocols.io.pa9dih6
  • 1Heinrich-Heine Universität Düsseldorf;
  • 2Institute for Synthetic Microbiology
  • Axmann Lab
  • CyanoWorld
Miriam D
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DOI: dx.doi.org/10.17504/protocols.io.pa9dih6
External link: https://www.addgene.org/protocols/create-glycerol-stock/
Protocol Citation: Miriam D, Anna Behle 2018. Preparing E. coli cryo cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.pa9dih6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 08, 2018
Last Modified: April 10, 2018
Protocol Integer ID: 11329
Keywords: cryoculture, cryogenic culture, E. coli, Escherichia coli, bacteria, storage, -80 °C
Abstract
This protocol describes how to prepare bacteria for further storage at -80 °C.
Guidelines
Always work under sterile conditions to avoid contamination.
Materials
MATERIALS
ReagentGlycerolCatalog #G5516
STEP MATERIALS
ReagentGlycerolCatalog #G5516
ReagentGlycerolCatalog #G5516
Protocol materials
ReagentGlycerolCatalog #G5516
ReagentGlycerolCatalog #G5516
ReagentGlycerolCatalog #G5516
ReagentGlycerolCatalog #G5516
Safety warnings
Always wear appropriate protection equipment while working with liquid nitrogen or dry ice.
Prepare sterile glycerol
Prepare sterile glycerol
Aliquot ~99% glycerol and autoclave.
You can also used filtered sterile glycerol.
ReagentGlycerolCatalog #G5516
Inoculate media
Inoculate media
Inoculate 3 mL of your desired media containing appropriate antibiotics with your desired Escherichia coli strain. Work under sterile conditions to avoid contamination.
Amount3 mL Media (i.e. LB)
Incubate cultures
Incubate cultures
Incubate your desired Escherichia coli cultures overnight until the culture reaches a exponential to stationary phase and a minimum absorbance of 1.0 and maximum absorbance of 5.0 at OD600.
Duration16:00:00 Incubation (overnight)
Centrifuge culture (optional)
Centrifuge culture (optional)
Centrifuge your well grown cultures for 10 minutes at 4,000 rpm.
Duration00:10:00 Centrifugation at 4,000 rpm
Discard supernatant (optional)
Discard supernatant (optional)
Discard the supernatant quickly. Do not disturb the pellet.
Resuspend (optional)
Resuspend (optional)
Resuspend your pellet in 800 µl of your desired media with specific antibiotics.
Amount800 µL media (i.e. LB)
Prepare cryo culture
Prepare cryo culture
Pipette 200 µl sterile glycerol in a cryo culture tube. Add 800 µl of your desired resuspended pellet/culture (20 % glycerol (v/v)).
You can also use glycerol concentrations up to 40 % (v/v). Most labs store bacteria in 15-25 % glycerol.
Amount200 µL glycerol
Amount800 µL bacteria culture
Freeze cryo culture
Freeze cryo culture
(Alternative: Freeze your cryo cultures with dry ice or liquid nitrogen.)
Store your cryo culture at -80 °C.