[Plasmid transformation of frozen and thawed bacteria]

Abstract

Plasmid DNA transformation efficiency depends on three essential factors: 1) the optimal regime of the recipients freezing-thawing; 2) the period of the recipients competence preservation; 3) individual sensitivity of microorganisms to freezing-thawing. It is demonstrated that plasmid DNA pMB9 activity indices are of maximal value during freezing at -70 degrees C or -196 degrees C and thawing at 42 degrees C. The short period of the competence, about 15 seconds, determines the rate of its infection. In this case it was achieved by mutual freezing-thawing of bacteria and DNA pMB9. The optimal yield of transformants is obtained in the following conditions: the concentration of bacteria - 1 - 5.10(9) cells/ml, the concentration of DNA pMB9 - 0.05--0.5 mcg/ml in the reaction mixture containing 0.5--1% of bactopeptone ("Spofa") and at pH 7.4--7.6.