Escherichia Coli Transformation Experiment Guide I4tcgwn
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1Published: 25 Jul 2017
This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source are credited
Escherichia Coli Transformation Experiment Guide
Caroline Green
Abstract
Escherichia Coli, also known as E. coli, is the most widely studied prokaryotic model organism,
and an important species in the field of biotechnology and microbiology, where it has served as
the host organism for the majority of work with recombinant DNA. Typically, E. coli expression is
the first choice for protein expression and protein product in E. coli is fast, convenient, well
established and always with high yields.
Experiment Principle
Plasmid DNA or recombinant DNA adhered to the surface of bacterial cells; 42 °C heat treatment
for a short time to promote the absorption of DNA and then cultivate a generation in the non-
selective medium; when the antibiotic gene on the plasmid expressed, it can be placed in the
medium containing antibiotics.
Experimental Materials
Plasmid DNA, recombinant DNA
Reagents, kits
LB medium, Distilled water, IPTG, X-gal, Ampicillin
Equipment
Vortex mixer, Micro-pipettes, Pipette tip, Centrifuge tube, Double-sided micro-centrifuge tube
rack, Dry air bath, Constant temperature water bath, Ice maker, Constant temperature shaker,
Petri dishes, Clean bench, Alcohol lights, Glass sticks, Constant temperature incubator
Operating Method
Adjust the temperature of the constant temperature water bath to 42°C1.
Geta tube (100 μl) of the competent bacteria from the -70 °C ultrafilter freezer and2.
2Published: 25 Jul 2017
This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source are credited
immediately melt with a finger and insert it into ice and ice for 5 to 10 minutes
Add 5 μl of the attached plasmid mixture (DNA content of no more than 100 ng), gently3.
shakeandplace on ice for 20 min
Gently shakeand insertinto the 42 °C water bath 1 ~ 2 min for heat shock, and then4.
quickly put back to the ice; put it aside for 3 ~ 5 min
Add 500 μl of LB medium (without antibiotics) to each of the tubes in a clean bench and5.
mix them gently onto a shaker of 37 °Cfor 1 h
In the clean bench, take the above conversion mixture 100-300 μl, respectively, to the6.
appropriate solid LB plateculture dish containing antibiotics; coat evenly with alcohol lamp
burned glass
If the carrier and host bacteria are suitable for blue-white screening, drop 40 μl of 2% X-7.
gal, 8 μl of 20% IPTG on the plate and coat evenlywith alcohol lamp burned glass
Mark on a coated dish and place in a 37 °Cincubator for 30 to 60 min until the liquid on the8.
surface penetrates into the culture medium and thenplace in the 37 °C incubator overnight
Spray 70% ethanol on the bacteria-contaminated table, dry the table, write an9.
experimental report
Collected by Creative BioMart.
Citation: Caroline Green Escherichia Coli Transformation Experiment Guide. protocols.io
dx.doi.org/10.17504/protocols.io.i4tcgwn
Published: 25 Jul 2017
