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Blue White Screenbing

This document describes a protocol for blue-white screening of bacterial colonies using X-Gal and IPTG plates. Blue-white screening is used to identify recombinant bacteria after cloning experiments. It relies on the enzymatic activity of β-galactosidase, which can metabolize X-Gal to form a blue product. The protocol details how to prepare X-Gal and IPTG solutions and incorporate them into agar plates, either during plate preparation or by adding to pre-made plates. Transformed bacterial colonies are then screened - white colonies indicate recombinant bacteria where the lacZ gene was disrupted during cloning.

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Payel Bose
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73 views2 pages

Blue White Screenbing

This document describes a protocol for blue-white screening of bacterial colonies using X-Gal and IPTG plates. Blue-white screening is used to identify recombinant bacteria after cloning experiments. It relies on the enzymatic activity of β-galactosidase, which can metabolize X-Gal to form a blue product. The protocol details how to prepare X-Gal and IPTG solutions and incorporate them into agar plates, either during plate preparation or by adding to pre-made plates. Transformed bacterial colonies are then screened - white colonies indicate recombinant bacteria where the lacZ gene was disrupted during cloning.

Uploaded by

Payel Bose
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Blue-White Screening of Bacterial Colonies Utilizing X-Gal and IPTG Plates

Introduction:-
Blue-white screening of bacterial colonies is a popular and effective molecular biology tool often
used to detect recombinant bacteria in cloning experiments. Central to this technique is the
enzymatic activity of β-galactosidase, a tetrameric enzyme encoded by the lacZ α gene in E. coli that
metabolizes lactose to form glucose and galactose. Alternatively, β-galactosidase can hydrolyze a
different substrate, X-Gal, resulting in 5-bromo-4-chloro-indoxyl, which dimerizes to form a blue
pigment. The phenomenon of α-complementation has made β-galactosidase a powerful molecular
cloning tool. In α-complementation, the deletion of a specific fragment of the lacZ ω gene in bacteria
resulting in an inactive β-galactosidase is resolved by the presence of a plasmid containing the
deleted fragment. In cloning, the plasmids routinely used contain a segment of the lacZ α gene,
while the E. coli host strain contain a lacZ ω deletion mutation. Thus, during transformation, when
bacteria containing the deletion take up the plasmid containing the deleted lacZ α segment,
functional β-galactosidase is produced. However, if the plasmid taken up by the bacteria is carrying a
piece of DNA (DNA of interest ligated into the plasmid using restriction sites during the cloning
process) that disrupts the lacZ α gene segment, recombinant bacteria result. Then, alpha
complementation cannot occur, and a functional β-galactosidase does not form. To perform blue-
white screening after transformation, X-Gal is added along with Isopropyl β-D1-
thiogalactopyranoside (IPTG), an inducer of lacZ ω gene expression. The blue colonies contain
bacteria with functional β-galactosidase, indicating the plasmid taken up during transformation did
not contain the DNA of interest. Conversely, the white colonies cannot metabolize X-Gal to produce
the blue color, because they do not produce functional β-galactosidase after taking up plasmid
carrying the inserted DNA and disrupting the lacZ α gene. These white colonies contain the
recombinant bacteria and should be selected (Figure 1). Here, we describe a protocol to perform
effective blue-white colony screening to select the recombinant bacteria carrying your DNA of
interest.

Materials :-
 X-Gal (GoldBio Catalog # X4281C)  Dimethylformamide (DMF)  dH2O  Isopropyl β-D-1-
thiogalactopyranoside, IPTG (GoldBio Catalog # I2481CE )  Screening antibiotic of choice  Agar
media (optional)  Plates

Method:-
1.Preparation of X-Gal and IPTG. X-Gal and IPTG can be incorporated into agar media before pouring
into plates or added onto pre-made plates.

I. Prepare 20 mg/ml X-Gal in DMF (see X-Gal Stock Solution Procedure).


II. Prepare 100mM IPTG solution in dH2O (see IPTG Stock Solution Procedure) or dilute from a
1M IPTG solution.

2. Screening on agar media containing IPTG and X-Gal (recommended)

I. Autoclave the growth media agar, then cool to 50°C.


II. Add 10 µl of 20 mg/ml X-Gal solution per 1 ml of media or 2 µl of 100 mg/ml X-Gal solution
per 1 ml of media.
III. Add 10 µl IPTG (100mM) per 1 ml of media for a final concentration of 1mM.
IV. Add the screening antibiotic.
V. Pour plates and allow them to cool to room temperature before use. This usually takes at
least 30 minutes.
VI. Spread transformed competent cells as desired.
3. Screening on pre-made agar plates lacking IPTG and X-Gal

I. Pour autoclaved growth media containing screening antibiotic on media plates and dry in a
laminar flow hood.
II. Add 40 µl 100mM IPTG and 120 µl X-Gal (20 mg/ml) to the surface of each plate and spread
over the entire surface. Note: The plate edges are difficult to spread evenly and may give
false positives. We advise picking colonies in the middle of the plate, if possible, for best
results.
III. Dry X-Gal/IPTG-coated media in a laminar flow hood for approximately 30 minutes before
use.
IV. Spread transformed competent cells and incubate inverted at either 37°C until blue colonies
form (usually ~24 hours)

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