MaizeGDB
jobs | upcoming events | sitemap
 | docs | bulk data | browse data | tools | login / register | links 
homeA popup window providing help for using the search form to the right  

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

Contents: Index | Libraries | Reports | Assembly | Annotation | Unigene | Search | Ordering | Protocols | FAQs

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)

N/A

N/A

Ampicillin (sodium salt) (25 mg/ml)

Sigma-Aldrich

A9518

LB agar

Prepared at Genome Center

N/A

Aliquot of frozen stock DH10Bä cells with pUC18 or pUC19 vectors

N/A

N/A

Deionized and distilled water (ddH2O)

Prepared at Genome Center

N/A

Terrific Broth w/salts (TBS) Freezing Media*

Prepared at Genome Center

N/A

glass beads (4mm)

VWR

26396-563

0.45 m m disposable Nalgene® Filter Unit

VWR

28199-097

Sterile, disposable Petri dish

Cole Palmer

KX-06139-02

100 % Glycerol

Roche

100 649

*TBS Freezing Media has 10% glycerol in solution

 

Procedure

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.


Return to Documentation Index | Return to Maize Gene Discovery Project index | Return to Homepage

home  

Be sure to cite us!

This page is HTML 4.01 valid!