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

✍ Scribed by H. Stompe; S. Michel; W. Mann; G. Richter

• Publisher: John Wiley and Sons
• Year: 2007
• Tongue: English
• Weight: 369 KB
• Volume: 22
• Category: Article
• ISSN: 0233-111X
• DOI: 10.1002/jobm.19820220708

✦ Synopsis

Abstract

Transfection by DNA 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 at the onset of late exponential growth (5 × 10^8^ cells/ml) and treating the latter with 0.05 M CaCl~2~. Hereafter, the microbes were suspended in 50 mM Tris‐HCl buffer (pH 7.2) and the concentration adjusted to 7 × 10^9^ cells/ml. T3 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 10^4^ p. f. u./μg DNA.

Preincubation of competent bacteria with T4 DNA at 0°C before the addition of T3 DNA reduced the number of infectious centers. However, if T3‐ and T4 DNA were added simultaneously no decrease of the transfection efficiency occurred. Calf thymus DNA was without influence on transfection.