Team:Washington/OVERNIGHT CULTURES

From 2013.igem.org


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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:

Reagent

Amount

Where to Find it

Broth (LB or TB)

3-5 mL (as needed)

Room temp or fridge stocks

Antibiotic(s)

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

-20 C Freezer or Refrigerator

Cells

1 colony
1 scrape from glycerol stock
10 uL liquid culture

Refrigerator
-80C Freezer
37C Incubator - Overnight culture

3. Gently mix, and place in 37C shaker-incubator overnight, with lid attached loosely for good aeration.
*Broth:
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.

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