Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.
Loading...
Date
2009-10
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
The efficiencies of different transformation methods of E. coli DH5α strain, induced by several cations like Mg2+, Mn2+, Rb+ and especially Ca2+, with or without polyethylene glycol (PEG) and dimethyl sulfoxide (DMSO) were compared using the two commonly used plasmid vectors pCAMBIA1201 and pBI121. The widely used calcium chloride (CaCl2) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl2 method were found to further augment the transformation efficiency (TR)E for both the vectors like repeated alternate cycles of heat shock, followed by immediate cold, at least up to the third cycle; replacement of the heat shock step by a single microwave pulse and even more by double microwave treatment and administration of combined heat shock-microwave treatments. The pre-treatment of CaCl2-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)E, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl2 method suggested in the present study may thus find use in more efficient E. coli transformation.
Description
Keywords
Calcium chloride, E. coli, Ethanol, Heat shock, Microwave, Plasmid DNA, Transformation efficiency
Citation
Roychoudhury Aryadeep, Basu Supratim, Sengupta Dibyendu N. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian Journal of Biochemistry & Biophysics. 2009 Oct; 46(5): 395-400.