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LABORATORY PROCEDURES FOR PLASMIDS

REFERENCE NO: PP/2003/03/03

TITLE: PRODUCTION OF THE MASTER STOCK AND THE DISTRIBUTION STOCK

INTRODUCTION

This document describes the basic procedure to be followed when producing the master stock and the distribution stock for the public collection.

Definitions

  • Master stock: The fully quality controlled stock of aliquots containing the 'original' material. These aliquots are not used for distribution. They are only subcultured in case the quality control of the distribution stock fails and a new distribution stock has to be produced.
  • Distribution stock: The fully quality controlled stock of aliquots which are used for distribution.

PROCEDURE 

  1. The host/plasmid subculture mentioned in PP/2003/03/01, paragraph 7, is divided in several aliquots to produce a fully quality controlled master and distribution stock. 
  2. The receptacles are labeled with a) the accession number and b) the date of preservation or a lot number, and preserved according to one of the following preservation methods:
  • cryopreservation of the host/plasmid combination below -70°C, using a cryoprotectant (e.g. glycerol, sucrose)
  • cryopreservation of the host/plasmid combination in liquid nitrogen, using a cryoprotectant
  • lyophilization of the host/plasmid combination, using a lyoprotectant

Detailed procedures for the different types of preservation methods should be available at the collection.

  1. Each deposit should whenever practical be maintained by at least two different preservation methods described above (cfr. WFCC Guidelines). Preservation of the plasmid DNA (preferably precipitated under ethanol) can also be applied as a second preservation method. A duplicate of the plasmid or the host/plasmid combination should be stored at a separate site (cfr. WFCC Guidelines). 

  1. Following preservation, one ampoule from the distribution stock is sent to the depositor, giving him the possibility to check the authenticity of the preserved material. 
  1. If the depositor found the preserved material not authentic, the collection requests new material from the depositor (PP/1998/02/02, paragraph 3 and 4). 
  1. If the depositor found the preserved material authentic, the master stock and the distribution batch are accepted. The catalogue entry is produced and the deposited material is made available to the public according to PP/1998/02/03. 

Furthermore, the plasmid should be submitted to the "maintenance plan" of the collection. This plan describes the scheme for periodic quality control tests of the preserved material. Several aspects determine the frequency of control tests: e.g. preservation method, turn-over of the plasmid, origin of replication of the plasmid, … 
  1.  When few aliquots of the fully quality controlled distribution stock are remaining*1, one aliquot is inoculated and subcultured in selective medium described by the depositor with a view to the production of a new distribution stock.

    *1: This implies an adequate stock control system.

  1. When the results are not found satisfactory, the other available aliquots from the fully quality controlled distribution stock are checked in the same way as described in paragraph 7. 
  1. When the results are found satisfactory, the host/plasmid subculture is divided in several aliquots to create a new distribution stock, taking into account paragraph 2 of this procedure. 
  1. The master stock is only approached in case all the available aliquots from the distribution stock failed to pass the quality control. In that case, one ampoule of the fully quality controlled master stock is resuscitated and subcultured in selective medium described by the depositor with a view to the production of a new distribution stock. 
  1. When the results are not found satisfactory, the other available aliquots from the fully quality controlled master stock are checked in the same way as described in paragraph 10. 
  1. When the results are found satisfactory, the host/plasmid subculture is divided in several aliquots to create a new distribution stock, taking into account paragraph 2 of this procedure. 
  1. The collection requests new material from the depositor, according to PP/1998/02/02, paragraph 3 and 4, in case all available aliquots from the fully quality controlled master stock failed to pass the quality control as described in paragraph 10.

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