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. Xgal solution, readytouse thermo fisher scientific. Oct 14, 20 x gal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. Page 2 general guidelines follow these guidelines when using subcloning efficiency dh5. This simple and rapid assay is suitable for expression. Xgal staining of mammary glands baylor college of medicine. Place plates upside down in incubator for 18 hours.
Cells were stained with xgal four days following transfection. This substrate yields a blue precipitate upon hydrolysis, making it suitable for use in immunoblotting or. Dgalactopyranoside is an inert chromogenic substrate for. The compound was synthesized by jerome horwitz and collaborators in detroit, mi, in 1964. Prepare a 20 mgml stock solution of xgal in n,n dimethylformamide dmf or dimethylsulfoxide dmso. Blue white screening of bacterial colonies using xgal. Pdf direct confocal acquisition of fluorescence from x. This protocol is for the preparation of x gal iptg lb agar plates for bluewhite colony screening. Dgalactopyranoside xgal stock solution at various concentrations. Preincubate glands in xgal solution without xgal for 1 hour at 37c. Bluewhite select screening reagent for selection of. Xgal 5bromo4chloro3indolyldgalactoside is a histochemical substrate for galactosidase. A previous report showed that sgal is a superior substitute for xgal in. For a more detailed protocol, contact technical service.
Direct confocal acquisition of fluorescence from xgal. Invitrogen xgal 1g pcr specific buffers fisher scientific. Thermo scientific xgal 5bromo4chloro3indolylbetadgalactopyranoside is an inert chromogenic substrate for betagalactosidase which hydrolyzes. The expression system for ease, yield, and functionality.
Untransformed gs115 was used as a control bottom right patch. Add the transformation reaction to the top agar after lawn cells, iptg, and xgal or bluogal have been added. Invitrogen xgal 100mg invitrogen xgal fisher scientific. One hundred percent of the ppicz btransformed colonies were blue, indicating that they contain the lacz gene. Let plate set on benchtop for 1015 minutes so that the solution absorbs into the agar. Add the transformation reaction to the top agar after lawn cells, iptg, and x gal or bluo gal have been added. X gal is supplied as white crystals and is soluble in dimethylformamide. Freestyle 293f cells were transfected with pcmvsport. 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. Dgalactopyranoside is an organic compound consisting of galactose linked to a substituted indole. This protocol is for the preparation of xgaliptg lb agar plates for bluewhite colony screening.
Component amount megax dh10b t1r electrocomp cells 5. We used pvitro2 neogfplacz vector invitrogen, carlsbad, ca, usa which. Generation of stable cell line by using chitosan as gene delivery. For each 35 mm well combine in polypropylene container. After transformation of the m vector, place transformed cells on ice. Xgal is useful for numerous histochemical and molecular biology applications, including detection of lacz activity in cells and tissues. It yields a dark blue precipitate at the site of enzymatic activity. The xgal staining assay kit is designed for sensitive and lowbackground in situ detection of cells transfected with vectors carrying the lacz gene. X gal is useful for numerous histochemical and molecular biology applications, including detection of lacz activity in cells and tissues.
Remove glands and fix in cold 4% pfa for 1 hour at rt. However, this procedure does not permit the direct confocal. Protein production in mammalian cells thermo fisher scientific. Typically used in conjunction with iptg for bluewhite screening via the lac operon. Add xgal or bluogal to the top agar to a final concentration of 50 gml and iptg to a final concentration of 1 mm. Direct confocal acquisition of fluorescence from xgal staining on thick tissue sections article pdf available in scientific reports 3. I tried to do x gal whole mount staining of murine embryos e15.
This simple and rapid assay is suitable for expression studies in cultured cells and tissues transfected in vivo. The x gal staining assay kit is designed for sensitive and lowbackground in situ detection of cells transfected with vectors carrying the lacz gene. Features and benefits intense color contrast for easy colony selection convenient, readytouse solution. Dgalactoside xgal in dmf 100 mgml ampicillin stock 50 mgml kanamycin 100 mm isopropyl. By contrast, only 19% of the colonies selected by histidine prototrophy were blue. Warm the selective plates in a 37c incubator for 30 minutes use 1 or 2 plates for each transformation. This protocol is for the preparation of xgaliptg lb agar plates for bluewhite.
Xgal and iptg table 1, sample 6, a reduction of about tenfold is seen in the transformation efficiency of our electroporation competent xl1 blue cells. These compounds are quite useful in screening for bacterial plasmid recombinants 1. Xgal inhibits the swarming of vibrio species sciencedirect. After obtaining a decent ish or even strong xgal signal in the whole mounts, i fix the embryos on or up to several days in 4% pfa, dehydrate them to 70% ethanol in pbs for a couple of days. Cells and reagent working together forhigh performance. Xgal also abbreviated bcig for 5bromo4chloro3indolyl. Cut fresh frozen sections and fix with cold formalin 4 c for 10 minutes. Xgal stock solution 25 mgml xgal 5bromo4chloro3indolylbdgalactopyranoside. Xgal is supplied as white crystals and is soluble in dimethylformamide. Store on ice until needed, or overnight at 4 c for maximum number of transformants transformation 1. Add all of the cellligationxgal solution to the lb plate and spread evenly with sterile spreading stick over plate.
Bacteria containing active galactosidase produce blue colonies when. Pdf direct confocal acquisition of fluorescence from xgal. Xgal staining assay kit, detection of cells genlantis. Taz construct are grown in m9 minimal medium without asp and spread on macconkey or x. Xgal staining is a common procedure used in the histochemical monitoring of gene expression by light microscopy. Components 40 mgml iptg 40 mgml xgal prepared in dmso principle. Gal assay kit from invitrogen provides the reagents required to quickly. Use it only with glass containers and glass pipettes. X gal obviously inhibited the swarming of all vibrio species used in this study. Galactosidase fluorescence probe with improved cellular. However, this procedure does not permit the direct confocal acquisition of images. To prepare 20x solution dissolve 20 mg of xgal in 1 ml dmf work in chemical fume hood. Spread xgal onto lb agar plates containing antibiotic, if desired.
Add x gal or bluo gal to the top agar to a final concentration of 50 gml and iptg to a final concentration of 1 mm. Invitrogen xgal 1g pcr specific buffers bioreagents. X gal is a widely used chromogenic substrate for galactosidase. The table below provides a general overview of the steps involved in the. Dry opened lb plates at room temperature under uv light for about 30 minutes. Pour 25 ml of prepared lb agar into each petri dish. Xgal is useful for numerous histochemical and molecular biology. This substrate yields a blue precipitate upon hydrolysis. General description sigmas bluewhite select screening reagent is a chromogenic substrate for.
Xgal is very expensive and should be used carefully. Hi, i do not know if this question was raised before, but i am looking for a good x gal antibody im working with murine embryos that works on paraffin sections. Get cells out of freezer, thaw on ice for 5 minutes 2. Step action 1 transfect cells with a plasmid expressing lacz. Preparation of xgaliptg lb agar plates for bluewhite. X gal 5bromo4chloro3indolyldgalactoside is a histochemical substrate for galactosidase. Xgal obviously inhibited the swarming of all vibrio species used in this study. After obtaining a decent ish or even strong x gal signal in the whole mounts, i fix the embryos on or up to several days in 4% pfa, dehydrate them to 70% ethanol in pbs for a couple of days. If you are including the puc19 control, make sure that you have one lb agar plate containing 100. Add all of the cellligation x gal solution to the lb plate and spread evenly with sterile spreading stick over plate. Xgal is dissolved at 20 mgml in dimethylformamide dmf and stored in darkcolored or aluminum foilwrapped tubes to protect from light. Invitrogen corporation 1600 faraday avenue carlsbad, ca 92008 t.
A typical stock concentration of xgal is 50mm 20mgml. Add the transformation reaction to the top agar after lawn cells, iptg, and x. This substrate yields a blue precipitate upon hydrolysis, making it suitable for use in immunoblotting or immunocytochemical assays. Dgalactopyranoside xgal stock solution at various concentrations typically used in conjunction with iptg for bluewhite. This protocol describes the preparation of a 5bromo4chloro3indolyl. To apply xgal directly to the top of agar plates for blue. V79020 and v79520 version k 10 november 2010 280104.
789 1257 451 540 413 474 1455 1296 1335 1363 634 1471 915 1013 616 1373 336 1321 30 1171 1211 1460 1116 1014 1099 760 1158 1399 469 1380 780 571 542 1285 1299 734 232 300 175 1384 1372