Question:
Which reagents are used for plasmid purification and colony screening?
The Protein Man Says:
There are a number of reagents that can be considered ideal for high throughput plasmid purification and colony screening. Chemical testing agents such as IPTG, X-Gal, X-Gluc and HP Ampicillin™ can all be used to successfully isolate DNA plasmids from the chromosomal DNA and cellular RNA and use them for research purposes.
Given the number of available reagents, how do you determine which among these testing agents will suit your intended purpose? To help you choose the most appropriate reagent for plasmid purification and colony screening, here is a brief description of the functions of each of these substances.
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a reagent used to induce the activity of ß-galactosidase, an enzyme that promotes lactose utilization. This specific reagent works by binding and inhibiting the lac repressor to allow the transcription of genes in the lac operon. Since it is not metabolized by E. Coli, IPTG concentrations usually remain constant in in vivo studies. IPTG is normally used with X-Gal to detect lac gene activity.
X-gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside) is an organic compound used in the study of molecular biology, genetics and other life sciences as a basis for the blue/white screening of recombinant clones and to detect the presence of ß-galactosidase, an enzyme which is commonly encoded by the lacZ gene. When cleaved by ß-galactosidase, X-Gal produces galactose and 5-bromo-4-chloro-3-hydroxyindole, which is then oxidized into 5, 5’-dibromo-4, 4’-dichloro-indigo, an insoluble blue product.
X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid) is a reagent that can be used to detect the presence of ß-glucuronidase (GUS), an enzyme produced by E. coli. X-Gluc is cleaved by the action of glucuronidase to produce glucuronic acid and chloro-bromoindigo, an intense blue precipitate. As such, this reagent can be quite useful in detecting GUS expression in plant cells and tissues as well as in detecting bacterial contamination in food and water samples. X-Gluc is commonly used for plasmid purification and colony screening since it gives fast and accurate results and is quite easy to transport and store.
HP Ampicillin™ is an antibitotic that inhibits the action of ß-lactamase in a stronger and more stable manner as compared to regular ampicillin. As such, it exclusively supports the growth of ampicillin-resistant colonies even after prolonged incubation and reduces the possibility of satellite colony formation.
Using the right reagent can help you isolate those precious DNA plasmids so that you can successfully proceed with your downstream experimentation.
Image By: Nathan Reading
