X-β-Gal, 100 mg
Molar mass (M) 408,6 g/mol
Melting point (mp) 230 °C
Storage temp. -20 °C
Transport temp. ambient temp.
CAS No. [7240-90-6]
Stock solution: 100 mg/ml (240 mM) in DMF, store at -20 °C in the dark
17,60 €/VE Campaign price!
Excl. btw | 100 mg Per VE
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X-β-Gal ≥99 %, BioScience Grade
Ideal for colorimetric detection of β-galactosidase activity, for instance during blue-/white selection.
Being a galacto pyranoside, IPTG inhibits the lac repressor, therefore leading to the induction the lac promotor. Subsequently, using the plasmid as matrix the C-terminal 146 amino acid fragment of the bacterial β-galactosidase is expressed, which functions as so called α-donor and complements the C-terminally deleted bacterial β-galactosidase. The then functional β-galactosidase restricts the galactoside X-Gal, resulting in a blue-coloured dye - therefore, these lac+ clones are coloured dark blue.
Since the C-terminal part of the α-donor is located upstream and the N-terminal part is located downstream of the multi cloning site of the plasmid, inserted DNA normally leads to frame shift or at least to major chain elongation, resulting in an unfunctional α-donor and no functional galactosidase - recombinant clones are white. The appearance of light blue clones either results form very short insertions or from the fact that in some plasmids the C-terminal fragment cloned upstream of the MCS is sufficient for α-complementation and restriction of a small amount of X-Gal.
Working solution: for blue/white selection of recombinant clones on agar plates use 0.3 μl stock solution per ml broth/agar.
|Signaalproduct||Precipitate (blue/white selection)|
- Tussentotaal: 0.00
Stock solutions (All solutions are stable for at least 1 year at 4 °C):
0.5 g NBT in 10 ml 70 % dimethylformamide;
0.5 g BCIP (p-toluidine salt) in 10 ml 100 % dimethylformamide.
Incubation buffer for alkaline phosphatase:
100 mM NaCl, 5 mM MgCl2,100 mM Tris (pH 9.5).
Fresh substrate solution: 66 μl NBT stock solution + 10 ml incubation buffer, mix well, add 33 μl BCIP stock solution. Use within 1 hour.
Blot development: Approx. 10 ml substrate solution per 15 x 15 cm2 membrane surface. Develop at room temperature until bands become visible (approx. 30 mins).
Reaction stop: Rinse with PBS/20 mM EDTA.
Dissolve 1 mg TMB in 0.1 ml dimethylsulfoxide (Art. No. 4720); add 9.9 ml of a 0.1 M sodium acetate solution (pH 6.0) (Powder: Art. No. 6779), filter and add H2O2 (Art. No. 8070) (final concentration 0.01 %).
Always prepare freshly!
Incubation 10-30 mins at room temperature (approx. 50 μl per microtitre well; finally add 50 μl 1 M H2SO4, Art. No. X873, per well).
Photometric quantitation at 450 nm.
Reference: Bos E.S. et al. (1981) J. Immunoassay 2, (3/4), 187.
|Appearance||white to off-white powder|
|Assay (HPLC)||≥99 %|
|Specific rotation [α]a (c=1 in DMF/H2O 1:1)||-62° ±2°|
|Water (KF)||≤0.2 %|
|DNases, RNases, Protease||none detected|