Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors.
The efficiencies of different transformation methods of E. coli DH5Qalpha train, 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.
Roychoudhury
Aryadeep
A
Department of Botany, Plant Molecular Biology and Biotechnology Laboratory, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India. aryadeep.rc@gmail.com
Basu
Supratim
S
Sengupta
Dibyendu N
DN
D002122:Calcium Chloride / Q000378:metabolism; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics*; D000431:Ethanol / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics*; D006358:Hot Temperature; D008872:Microwaves; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial / Q000187:drug effects / Q000528:radiation effects
D000431:Ethanol; D002122:Calcium Chloride