Jan 01, 2020
Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids
- 1Addgene
External link: https://www.addgene.org/protocols/create-glycerol-stock/
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2020. Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids. protocols.io https://dx.doi.org/10.17504/protocols.io.4g5gty6
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: June 21, 2019
Last Modified: August 18, 2020
Protocol Integer ID: 24829
Abstract
This protocol is for creating bacterial glycerol stocks for long-term storage of plasmids. To see the full abstract and other resources, visit the Addgene protocol page.
Guidelines
Tips and FAQ
- The optimal concentration of long-term glycerol storage is unknown. Most labs store bacteria in 15-25% glycerol.
- You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning, when you retrieve your liquid bacterial culture, take 500 µL of culture to make your glycerol stock before you begin your plasmid mini-prep.
- Try not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely and will improve the shelf life.
- It is very important that you shake the glycerol before freezing (5-6 times). Make sure that you see one uniform solution, and there are no layers present.
- Be sure to label both the lid and the tube of a glycerol stock before you place the sample at -80 °C . Frozen tubes are hard to write on and samples stored for long periods at -80 °C can lose labels stuck to tube!
After you have bacterial growth, add 500 µL of the overnight culture to 500 µL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.
Note
Notes:
- Make the 50% glycerol solution by diluting 100% glycerol in dH20.
- Snap top tubes are not recommended as they can open unexpectedly at -80 °C .
Freeze the glycerol stock tube at -80 °C . The stock is now stable for years, as long as it is kept at -80 °C . Subsequent freeze and thaw cycles reduce shelf life.
To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
Grow your bacteria overnight at the appropriate temperature. Growth conditions, including copy number and growth temperature, can be found on your plasmid's information page. The next day you will be able to start an overnight culture for plasmid DNA prep the following day.