QUALITY CONTROL CHECK OF PLASMID DNA

LABORATORY PROCEDURES FOR PLASMIDS

REFERENCE NO: PP/2003/03/02

TITLE: QUALITY CONTROL CHECK OF PLASMID DNA

INTRODUCTION

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

PROCEDURE

Plasmid DNA is prepared from the subculture mentioned in PP/2003/03/01, paragraph 7, based on a published plasmid isolation procedure, e.g. Birnboim et al. (1979), Ish-Horowicz et al. (1981), Rohde (1995).

The plasmid length should be verified by one of the following methods:
a) Determination of the molecular weight of the covalently closed circle (ccc) DNA (Meyers et al. (1976), Southern (1979)). It is necessary to use at least two ccc plasmid standards with known molecular weights close to the plasmid length to be determined.

b) Analysis of the restriction site pattern by agarose gel electrophoresis after single and (when possible) double restriction enzyme digestion.

The following criteria have to be checked:

In case 2a) has been carried out

The plasmid size estimated from the molecular weight of the ccc DNA must match the depositor's data.

In case 2b) has been carried out

- The plasmid size estimated from linearized DNA must match the depositor's data.

- Where two or more restriction sites are known, the presence and the location of at least two restriction sites must be confirmed.

If the criteria described above are not fulfilled, the collection contacts the depositor requesting new material as described in PP/1998/02/02, paragraph 3 and 4.

If the criteria described above are fulfilled, a master stock and a distribution stock are produced according to PP/2003/03/03.

References

Birnboim et al. (1979). A rapid alkaline extraction procedure for screening recombinant plasmide DNA. Nucleic Acids Research 7, 1513-1519

Ish-Horowicz et al. (1981). Rapid and efficient cosmid cloning. Nucleic Acids Research 9, 2989-2998

Meyers et al. (1976). Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. Journal of Bacteriology 127, 1529-1537

Rohde (1995). Technical Information Sheet No. 12: Plasmid isolation from Bacteria: some fast procedures. World Journal of Microbiology & Biotechnology 11, 367-369

Southern (1979). Measurement of DNA length by gel electrophoresis. Analytical Biochemistry 100, 319-323

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