Archive \ Volume.2 2011 Issue 2

Optimum range of plasmid supercoiled DNA for preparation of competent Top 10 E. coli

Muhammad Tahir Majeed , Khalid Hussain , Mamoona Noreen , Mateen Izhar
Abstract

Objectives: In-house preparation of chemically competent and electrocompetent Top 10 E. coli is not only economical but meets the needs for most of the molecular cloning work. For such transformations an optimum range of plasmid supercoiled DNA is needed. Therefore, the present study describes the modification of two protocols for the preparation of such cells, and optimization of the amount of plasmid supercoiled DNA required for better efficiency. Materials and methods: As most of the available protocols to render bacterial cells competent need special media or chemicals and are time consuming, the methods from Helen Donis-Keller Laboratory Manual of Washington University in St. Louis and Goldberg Laboratory Standard Protocols of the United States Department of Agriculture have been used after meticulous selection and with few modifications for preparing chemically competent and electrocompetent Top 10 E. coli, respectively. The transformation was carried out using pUC19 supercoiled plasmid DNA. Results: The transformation efficiencies of chemically competent and electrocompetent Top 10 E. coli were found to be 1.1 x 106 and 7.88 x 107 tranformants/μg of DNA, respectively. Such efficiencies are slightly higher than the required (105-106 transformants/μg DNA) for most of the cloning experimentation.