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Title: Analysis of comparative efficiencies of different transformation methods of <i style="">E. coli</i> using two common plasmid vectors
Authors: Roychoudhury, Aryadeep
Basu, Supratim
Sengupta, Dibyendu N
Keywords: Calcium chloride
<i style="">E. coli</i>
Heat shock
Plasmid DNA
Transformation efficiency
Issue Date: Oct-2009
Publisher: CSIR
Abstract: The efficiencies of different transformation methods of <i>E. coli </i>DH5<sub>α </sub>strain, induced by several cations like<b style=""> </b>Mg<sup>2+</sup>, Mn<sup>2+</sup>, Rb<sup>+</sup> and especially Ca<sup>2+</sup>, 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 (CaCl<sub>2</sub>) method appeared to be the most efficient procedure, while rubidium chloride (RbCl) method was the least effective. The improvements in the classical CaCl<sub>2</sub> method were found to further augment the transformation efficiency (TR)<sub>E</sub> 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 CaCl<sub>2</sub>-competent cells with 5% (v/v) ethanol, accompanied by single heat shock also triggered the (TR)<sub>E</sub>, which was further enhanced, when combined heat shock-microwave was applied. The minor alterations or improved approaches in CaCl<sub>2</sub> method suggested in the present study may thus find use in more efficient <i>E. coli</i> transformation.
Description: 395-400
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.46(5) [October 2009]

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