1,044
edits
Changes
Jump to navigation
Jump to search
Line 1:
Line 1: − + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
added S-Gal section
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside:
=S-Gal=
S-Gal is a trademark product of Sigma Aldrich for the compound 3,4-cyclohexenoesculetin-
β-D-galactopyranoside
[[Image:sgal.tiff|400px|left]]
Image from Heuermann and Cosgrove PMID 11355350
Reagents ordered in February 2009
0F5879-100G, $35.54, AMMONIUM IRON(III) CITRATE, REAGENT GRADE
S7313-1G, $385.60, S-GAL(R) SODIUM SALT
==S-Gal staining conditions==
'''Bone Marrow Cells for use in MRI, From Bengtsson et al. in NHMFL reports'''
BMCs were harvested from C57 and ROSA26 mice and re-suspended with 1 mg/ml S-Gal (Sigma) and 0.5 mg/ml FAC
(Sigma) in 1ml PBS, 0.5 mg/ml FAC in 1 ml PBS or 1 ml PBS in microcentrifuge tubes and incubated at 37°C for 2hrs.
BMCs were then washed twice with PBS to remove fcaps to remove large aggregations of S-Gal and cells.
'''In Mouse Embryos from Kishigami et al PMID 16419090'''
(In brief, dissected embryos were fixed with 4% paraformaldehyde overnight, subse- quently soaked with 20% sucrose in PBS, and embedded in Optimal Cutting Temperature (O.C.T.) in liquid nitro- gen. Fourteen-lm sections were cut, washed with PBS at room temperature, soaked with 100 mM sodium phos- phate buffer (pH 7.5), 0.1% sodium deoxycholate, 0.2% Nonidet P-40, 2 mM magnesium chloride, 5 mM potas- sium ferricyanide, and 5 mM potassium ferrocyanide for 10 min at room temperature. Sections were subse- quently stained with 1 mg/ml of X-gal or S-gal at 378C. An S-gal-stained section was immediately used for in situ hybridization according to the nonradioactive protocol (Lindahl et al., 1997).
'''For detecting plasmids on agar plates, from Heuermann and Cosgrove PMID 11355350'''
For chemical analysis, ferric ammonium citrate, S-Gal, and IPTG were added to 50-mL aliquots LB broth or deionized water at final concentrations of 500, 300, and 30 µg/mL, respectively (all from Sigma). S-Gal dye was initially dissolved in N,N-dimethylformamide (DMF) (Sigma) at a concentration of 200 mg/mL and was added to water or medium for analysis at the concentration stated above before autoclaving or microwaving. For this part of the evaluation, a stock solution of S-Gal in DMF was used for convenience and for the consistent addition of S-Gal to media or aqueous solu- tions. S-Gal, determined empirically to be approximately 70% more soluble than X-gal at room temperature (data not shown), is suitable for the blended-medium product but re- quires dissolving in DMF for the preparation of more concen- trated solutions such as the stock solution described here. IPTG was added to the water or medium from an aqueous stock of 100 mg/mL. Ferric ammonium citrate was prepared as an aqueous stock solution of 200 mg/mL.
S-Gal, IPTG and ferric ammonium citrate are dry-blended with standard LB agar at the final concentrations stated above. The
powdered blend was suspended in deionized water and autoclaved as previously described. For the preparation of microwaved medium, the suspended blend was heated until boiling, followed by swirling to allow the remaining agar component to go into solution. In this case, the antibiotic ampicillin was added after autoclaving (100 mg/mL).
=X-Gal=
5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (MW 408.63):
[[Image:xgal.gif|400px|left]]
[[Image:xgal.gif|400px|left]]