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Project Documentation & Protocols: Maize Gene Discovery Project: ESTs: Protocols: Colony Growth

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Colony Growth

EST Sequencing: Colony Growth

General Description: The source DNA is obtained from various labs and individuals. The sequencing libraries are received as frozen stock DH10Bä cells with pUC18 or pUC19 vectors. The source DNA is then plated onto agar plates with ampicillin (final concentration of 100 m g/ml) and incubated in preparation for picking. This protocol describes the procedures for media preparation, plating and incubation of an EST library.


Materials & Reagents:
Material / Reagent Vendor Stock #

Stock library dilution (freezing media, sample DNA)



Ampicillin (sodium salt) (25 mg/ml)



LB agar

Prepared at Genome Center


Aliquot of frozen stock DH10Bä cells with pUC18 or pUC19 vectors



Deionized and distilled water (ddH2O)

Prepared at Genome Center


Terrific Broth w/salts (TBS) Freezing Media*

Prepared at Genome Center


glass beads (4mm)



0.45 m m disposable Nalgene® Filter Unit



Sterile, disposable Petri dish

Cole Palmer


100 % Glycerol


100 649

*TBS Freezing Media has 10% glycerol in solution



Preparation of TB Freezing Buffer

  1. Add 50 ml TB salts (see below) to 450 ml TB (see below) and mix thoroughly.
  2. Extract 50 ml of TBS solution and add 50 ml of 100% glycerol stock solution to TBS and mix thoroughly.
  3. The resulting volume should be 500 ml with 10% glycerol in solution.
  4. Filter sterilize using a 0.45 m m disposable Nalgene® Filter Unit.

Preparation of Terrific Broth (TB)
Prepared and supplied at Stanford Genome Technology Center

  1. Add 6 g bacto-tryptone, 12 g bacto-yeast extract and 2 ml glycerol to 450 ml ddH2O.
  2. Mix thoroughly and autoclave.

Preparation of TB Salts
Prepared and supplied at Stanford Genome Technology Center

  1. Add 1.2 g Potassium phosphate (Monobasic) and 8.2 g Potassium phosphate (Dibasic) to 50 ml ddH2O.
  2. Mix thoroughly and autoclave.

Agar Plate Preparation

  1. Put 500 ml jar of LB agar in microwave on low power until fully liquefied.
  2. Let glass jar cool enough to be held; do not overcool or agar will solidify.
  3. Add 2 ml (25 mg/ml) ampicillin (or carbenicillin) while still warm. (Note: Final concentration of ampicillin in agar should be 100m g/ml.)
  4. Pour evenly into Petri dishes and let cool with tops on.

Culture and Incubation

  1. Prepare a dilution of the stock library to yield 100-150 colonies per 100 ml.
  2. Add approximately one dozen beads to each agar plate.
  3. Using pipet, add 100 ml of library dilution into center of each plate.
  4. Cover and shake plate for approximately 30 seconds to spread cells across surface of agar.
  5. Dump out beads and re-cover.
  6. Label agar plate cultures according to sample type.
  7. Let set right side up for 15 minutes on counter top.
  8. Place inverted plates in incubator at 37 °C for 20 hours.
  9. After removing from incubator, pick colonies or store at 4 °C for later use.

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