Iptg is also used to induce the expression of cloned genes that are under control of the lac operon. Xgal staining assay kit, detection of cells genlantis. Avoid raising and breathing dust, and provide adequate ventilation. Invitrogen xgal 100mg invitrogen xgal fisher scientific. Pdf direct confocal acquisition of fluorescence from x. All other chemicals and materials were obtained from the sigmaaldrich. Direct confocal acquisition of fluorescence from xgal. Galactosidase reporter gene staining kit sigmaaldrich. Galactosidase staining kit utilizes xgal as the substrate and lacz gene as the reporter for testing the efficiency of expression vector mediated gene transfer and for studying the regulation of promoters of genes. Bacteria containing active galactosidase produce blue colonies when. Xgal with iptg is essential for bluewhite screening. A typical stock concentration of xgal is 50mm 20mgml. Remove glands and fix in cold 4% pfa for 1 hour at rt.
Methods and material for containment and cleaning up. Xgal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. Dgalactopyranoside xgal stock solution at various concentrations typically used in conjunction with iptg for bluewhite screening via the lac operon. Routine subcloning into plasmid vectors bluewhite screening of transformants on selective plates containing bluogal or xgal highquality plasmid preparation transformation of large plasmids up to 30 kb. Thermo scientific xgal 5bromo4chloro3indolylbetadgalactopyranoside is an inert chromogenic substrate for betagalactosidase which hydrolyzes. The substrate used in this kit is x gal 5bromo4chloro3indol yl. Alternatively, xgal staining is also used to analyze protein expression pattern. Betagalactosidase staining kit k802 biovision, inc. Preincubate glands in xgal solution without xgal for 1 hour at 37c.
Component amount megax dh10b t1r electrocomp cells 5. Dgalactopyranoside which results in an indigo bluecolored staining of transfected cells and tissues. Incubation at 37c for 1 hour is not required after addition. This protocol describes the preparation of a 5bromo4chloro3indolyl. Cells should be at or less than 5080% confluence at the time of assay. Oz biosciences xgal staining kit allows to visualize gal expression through hydrolysis of the xgal 5bromo4chloro3indoylbetadgalactopyranoside substrate yielding blue precipitates. For each 35 mm well combine in polypropylene container.
Dry opened lb plates at room temperature under uv light for about 30 minutes. This substrate yields a blue precipitate upon hydrolysis, making it suitable for use in immunoblotting or immunocytochemical assays. This simple and rapid assay is suitable for expression studies in cultured cells and tissues transfected in vivo. Xgal is supplied as white crystals and is soluble in dimethylformamide. Use it only with glass containers and glass pipettes. Hi, i do not know if this question was raised before, but i am looking for a good xgal antibody im working with murine embryos that works on paraffin sections. After transformation of the m vector, place transformed cells on ice. Cells transformed with vectors containing recombinant. Preparation of x gal iptg lb agar plates for bluewhite. In this method, genetically modified animals that express betagalactosidase under the control of certain regulatory elements will be used to reveal the expression. Overview and assessment of the histochemical methods and.
Xgal staining on adult mouse brain sections bioprotocol. It is simple and routinely used to detect expression pattern of any proteins of interest in vivo. Information is provided elsewhere in the manual if you need help with any of the steps. To achieve optimal performance of bluo gal the following conditions are necessary when using bluo gal on bacterial plates. I tried to do xgal whole mount staining of murine embryos e15. Of 206 enterobacteriaceae strains able to hydrolyze xgal, 194 94. Invitrogen xgal 1g pcr specific buffers bioreagents.
This test provides a method for determining the percentage of transfected cells that are expressing lac z or for microscopically visualizing reporter gene expression in. Store at 4c or at room temperature in the original vial. Divide into 200 l aliquots and freeze at 20 degrees c. To prepare 20x solution dissolve 20 mg of xgal in 1 ml dmf work in chemical fume hood. Pdf xgal staining is a rapid and convenient histochemical technique used to detect reporter.
Pour 25 ml of prepared lb agar into each petri dish. Lacz is one of the most frequently reporter gene used in transfection experiments. The xgal staining assay kit is designed for sensitive and lowbackground in situ detection of cells transfected with vectors carrying the lacz gene. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of xgal. This protocol is for the preparation of x gal iptg lb agar plates for bluewhite colony screening. The compound was synthesized by jerome horwitz and collaborators in detroit, mi, in 1964. If xgal staining is to be avoided for example, if the fixation procedure is not compatible with other labelings or if the blue precipitate interferes with optical measurements. Xgal is very expensive and should be used carefully. Xgal staining see also the protocol in current protocols in molecular biology 1. Please login or register for free to view full text. Gal staining kit from invitrogen provides reagents required to determine the percentage of cells transfected with a plasmid expressing lacz.
Realtime imaging of senescence in tumors with dna damage. If electrocompetent cells are being used, place the electroporation chamber on ice. To apply xgal directly to the top of agar plates for bluewhite screening, apply 40. The substrate used in this kit is xgal 5bromo4chloro3indol yl. Invitrogen xgal 1g pcr specific buffers fisher scientific. Precast gels and is intended to supplement the gel instruction cards im6000 to im6008 supplied with the precast gels. The xgal is in dimethylformamide, and bathing the cells in a nice solution of that cant be good if i had to find a way to save it, i would be more tempted to swirl some lb over them and replate a small bit of the broth on a new xgaliptg plate, or replica plate. After inducing senescence, cells were fixed with 4% paraformaldehyde for 5 min and then stained with xgal staining solution 40 mm citric acidsodium phosphate buffer, 5 mm k 4. Dgalactopyranoside is an organic compound consisting of galactose linked to a substituted indole. Transfer to a chemical waste container for disposal in accordance with local regulations. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl galactoside. The lacz encoded protein betagalactosidase, betagal, is very stable, resistant to proteolytic degradation and easily. This substrate yields a blue precipitate upon hydrolysis. X gal is supplied as white crystals and is soluble in dimethylformamide.
Lacz is a bacterial gene often used as a reporter construct in eukaryotic transfection. Accidental release measures contain spill and collect, as appropriate. The bluewhite screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vectorbased molecular cloning experiments. Xgal is expensive stuff, but one purchase should give you enough aliquots for many labs. Xgal also abbreviated bcig for 5bromo4chloro3indolyl. Its sensitivity for staining tissue sections is similar to that of xgal. One shot top10f competent cells thermo fisher scientific. Xgal stock solution 25 mgml xgal 5bromo4chloro3indolylbdgalactopyranoside. Cell experiment cell lines human embryonic kidney 293t cells was supplied by the institute of cell biology shanghai, china. Kit components fixative solution 1x xgal 150 mg, lyophilized. The competent cells should be added to top agar after lawn cells, iptg and bluo gal or x gal have been added. Xgal is useful for numerous histochemical and molecular biology applications, including detection of lacz activity in cells and tissues.
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