Abstract
This protocol describes a quick and efficient method to make competent E. coli cells for transformation using rubidium chloride. Commercial competent cells are expensive and this protocol provides a cheaper alternative to them.
Keywords: Competent cells, E. coli, Transformation efficiency, TOP10, DH5α
Background
The success of gene cloning is highly dependent on the transformation efficiency of bacterial cells. The efficiency can be artificially improved by treating the cells with chemicals or electric pulses. Several protocols are available to prepare competent E. coli cells, however, they are usually long, laborious, and show inconsistency in competence. The protocol by Green and Rogers (2013) overcomes these downsides and allows the preparation of highly competent cells (~106-108 CFU/µg DNA). While other protocols require cells to be grown at low temperature (19-22 °C), this protocol involves growing cells at 37 °C. Thus, the cells grow faster and reach log phase within 4 h as compared to 18-24 h. This protocol is highly reproducible.
Materials and Reagents
Equipment
Procedure
Notes
This protocol is highly reproducible if you:
Recipes
Acknowledgments
This protocol is adapted from Green and Rogers (2013). I would like to thank Bergmann lab members for fine-tuning this protocol. The authors declare that there are no conflicts of interest.
References
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Thanks for the question! There's three main reasons:1) You want to select colonies that are growing fast and look uniform. Sometimes colonies can be of different sizes reflecting possible mutations in the cell pool. Select the big ones.2) The whole process happens without antibiotic selection and cultures could get contaminated with airborne bacteria (take into consideration that you are doing this in a lab where the same bacteria is present with plasmids providing antibiotic resistances). You want to select bacterias that can be tracked back to the plate with the streak. 3) E.coli might spontaneously develop spectinomycin resistance. When plated on spec plates, very small colonies will appear. If you use pool of cells, you might end up enriching in these contaminants.