Storing Bacteria

How do you save your work in a molecular biology lab? Following cloning, your DNA constructs (e.g. constructed plasmids), should be stored in the -20°C freezer. This is OK, but DNA has a shelf life (~many years) and does degrade at a slow rate. One preferred way to save your work is to transform your DNA into a cloning strain of E. coli and to store that cell line in the -80°C freezer. You can always streak these cells out on appropriate selective plates, and then subsequently perform a plasmid prep to extract the plasmid, i.e. open your work - to extend the file versioning analogy.

Protocol to prepare a bacterial glycerol stock

1. Select several glycerol stock tubes (twist on caps) from drawer beneath PCR supplies drawer

2. Centrifuge 5 ml culture(s) in tabletop centrifuge at max RPMs for 5 minutes

3. Decant supernatant near flame to ensure sterile conditions

4. Resuspend Pellet using 750 ul appropriate media (LB for E. coli)

5. Take 750 ul of resuspended cell media and add to glycerol stock tube

6. Add 750 ul 50% glycerol solution to glycerol stock tube (final glycerol concentration of 25% glycerol)

7. Vortex glycerol stock tube

8. Store in -80°C freezer

Notes

Glycerol stock tubes should be labeled with the following information: Strain, initials of scientist, date of preparation, strain traits (or plasmids carried), number of strain if in a series or progression, media used, antibiotic resistance of cells.

Example: DH10B, ACR, 01/31/18, pFTV-RBS-ackA-mRFP1, LB, CmR

Streaking out your cells

1. 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. Repeated freeze-thaw cycles damage and kill the cells. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely and will improve the shelf life.

2. 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.