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LABORATORY PROCEDURES FOR PLASMIDS

REFERENCE NO: PP/2003/03/01

TITLE: QUALITY CONTROL CHECK OF HOST/PLASMID COMBINATIONS

INTRODUCTION 

This document describes the basic procedure to be followed when checking the quality of the host/plasmid combination upon arrival of a new plasmid for the public collection. 

PROCEDURE 

  1. Upon receiving the plasmid, it is stored as appropriate (e.g.: if the material arrives frozen, ampoules have to be stored at minimum -80°C) until culturing can be started. 
  1. If the plasmid arrives as pure DNA, the appropriate host bacterium is first transformed with this DNA. 
  1. The host/plasmid combination is streaked on sterile, selective solid medium described by the depositor. The collection should have separate procedures on the aspects of preparation and storage of media at its disposal.
  1. After incubation during minimum 12 hours at the appropriate temperature, the host/plasmid colonies are examinated visually and/or microscopically. 
  1. The following criteria have to be checked: 
  • Viability

The host/plasmid combination must be recoverable under specific selection for the plasmid 

  • Morphology / Purity

The texture, the size and the opacity of the colonies should be in accordance with the characteristics of the host bacterium;

Homogeneity of the colonies is required;

Growth of (slow growing) contaminants (e.g. other bacteria, fungi) is not allowed.

  1. If the visual examination does not fulfil the criteria described above, the collection contacts the depositor requesting new material as described in PP/1998/02/02, paragraph 3 and 4. 
  1. If the visual examination comes up to the criteria described above, a single colony is subcultured in selective medium described by the depositor, with a view to the quality control of the plasmid DNA (PP/2003/03/02) and to the preparation of the master stock and the distribution stock (PP/2003/03/03).

Guidelines prepared for CABRI by BCCM/LMBP in cooperation with DSMZ and NCCB, 7 May 1998 ; updated dec. 2003
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