Preparation of Overnight Cultures

Use TB for making glycerol stocks and TB for minipreps.

  1. Obtain a 14 mL Sterile Polypropylene Round-Bottom Tube.
  2. Add ~ 3-5 mLs of an appropriate broth (either LB or TB)* see tips
  3. Add an appropriate antibiotic if necessary and set this tube safely aside
    1. Amp (1:1000)
    2. Chlor (1:1000)
    3. Kan (1:1000)
  4. Incubate overnight cultures @ 37oC, overnight in the iGEM labeled shaker

General Purpose Short Protocol:

Set-up Time: 5 min
Total Time: 12 - 24 hours (18 hours for best plasmid production)

1.  Label a 15 mL culture tube with strain/gene, antibiotic, and date.
2.  Add the following to tube as appropriate:



Where to Find it

Broth (LB or TB)

3-5 mL (as needed)

Room temp or fridge stocks


1 uL/mL - Amp/Carb
1 uL/mL - Kan
1 uL/mL - Cm
1 uL/mL - Sp

-20 C Freezer or Refrigerator


1 colony
1 scrape from glycerol stock
10 uL liquid culture

-80C Freezer
37C Incubator - Overnight culture

3. Gently mix, and place in 37C shaker-incubator overnight, with lid attached loosely for good aeration.
The type of broth used varies by the application.  If we are growing up cells to miniprep, we typically use TB to get a higher OD of cells, though LB works well for any other applications.  Broth that is pre-mixed with antibiotics should be stored in the refrigerator for no longer than 2 months.
2013 UW iGEM is using LB from capsules which has gelatin that will clog purification columns. The TB should be made from powder.