Charakterisierung eines CaCl₂‐abhängigen Transfektionssystems von Escherichia coli und T3‐Phagen‐DNS

Abstract

Transfection by DKA isolated from bacteriophage T3 was studied using Escherichia coli 921/0 as host. The following conditions were found optimal:Competent E . coli 921/0 were obtained by harvesting the bacteria a t the onset of late exponential growth ( 5 x lo8 cells/ml) and treating the latter with 0 . 0 5~ CaCI,. Hereafter, the microbes were suspended in 50 mM Tris-HCI buffer (pH 7.2) and the concentration adjusted to 7 x lo9 cells/mI. T 3 DNA was added and the suspension kept at 0 "C for 15 min. Determination of the number of infectious centers was then carried out in the usual way. The efficiency of transfection under these conditions amounted to lo4 p. f. u./Fg DNA.Preincubation of competent bacteria with T4 DNA a t 0 "C before the addition of T3 DNA reduced the number of infectious centers. However, if T3-and T4 DKA were added simultaneously no decrease of the transfection efficiency occurred. Calf thymus DNA was without influence on transfection.