M. Ximena Anleu Gil —BIO-PROTOCOL

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Diego Wengier 1 protocol

Nidhi Sharma HHMI/Stanford University
2 protocols

M. Ximena Anleu Gil

Department of Biology, Stanford University, USA, USA,

Research focus

Microbiology

1 Author merit
Reviewer merit
Editor merit

Education

Bachelor of Arts, Swarthmore College, USA, 2015

Current position

Lab Technician, Biology, Stanford , USA

Publications

Raissig, M. T., Matos, J. L., Anleu Gil, M. X., Kornfeld, A., Bettadapur, A., Abrash, E., Allison, H. R., Badgley, G., Vogel, J. P., Berry, J. A. and Bergmann, D. C. (2017). Mobile MUTE specifies subsidiary cells to build physiologically improved grass stomata. Science 355(6330): 1215-1218.

1 Protocol published

A Quick and Easy Method for Making Competent Escherichia coli Cells for Transformation Using Rubidium Chloride

Authors: Nidhi Sharma, M. Ximena Anleu Gil and Diego Wengier, date: 11/05/2017, view: 11308, Q&A: 1

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.

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I was wondering why we should pick a single colony for making competent cells? Is there any difference between single colony and a mixture of E. coli culture? (Perhaps because of reproducibility? I guess.)

11/26/2019 10:10:24 AM Reply

Diego Wengier

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.

2019-11-26 05:48:25