14 NEED TO KNOW IPTG FAQS AND PROTOCOLS
IPTG is a molecular biology reagent utilized in cloning experiments. This compound is used as a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lacoperon. Unlike allolactose, the IPTG sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant.
QUESTION: What are the alternative names?
ANSWER: Isopropyl β-D-1-thiogalactopyranoside, IPTG, Isopropyl Thiogalactoside
QUESTION: WHAT IS THE DETAILED DEFINITION OF IPTG?
ANSWER: IPTG induces the transcription of the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. One advantage of IPTG for in vivo studies is that since it cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-controlled genes, IPTG is not a variable in the experiment.
IPTG intake is independent on the action of lactose permease, since other transport pathways are also involved.In cloning experiments, colonies that have been transformed with the recombinant plasmid rather than a non-recombinant need to be identified. X-GAL is a substance that can be metabolized by beta-galactosidase to produce a blue product. Thus cells expressing beta-galactosidase grown in the presence of X-GAL and IPTG (to induce the expression) will turn blue. Where a DNA fragment has been inserted into the LacZ (one of the genes for beta-galactosidase) there will be no action upon X-gal and the cells will not turn blue, thus identifying the cells that carry recombinant plasmid rather than non-recombinant plasmid.
1) From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 30°C (or 37C) in 1-2ml LB+AMP (or other selection) in a 15ml snap cap tube on a rotator or shaker.
2) Dilute 1:50 (1:100 if 37°C O/N) in 2ml LB+AMP and grow 3-4 hours at 37°C in 15ml snap cap tube in a rotator.
4) Prepare 1ml LB+AMP+1mM IPTG in a 15ml conical and prewarm to 37°C about 10min before use.
To be continued..
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