ONPG Assay

(for assessing the level of chimerism in LacZ+ founders)


Solutions


100X Magnesium Chloride Solution (1 ml)

	100 ul	1M MgCl2
	281 ul	12.7 M Beta-Mercaptoethanol
	619 ul 	diH2O


0.1 M Sodium Phosphate Solution @ pH 7.5 (50 ml)

	4.1 ml 	1M Na2HPO4
	0.9 ml	1M NaH2PO4
	45  ml 	diH20


1M Sodium Carbonate Solution  (1 M)

	10.6 g Na2CO3
		  diH2O to volume

Beta-Galactosidase Solution
	
	3000 units/ml in 0.1M NaPO4 pH 7.5
		dilute 1ul : 1000ul just prior to use to equal 3.0 units/ml

1X ONPG Solution (13.27 mM)

	4mg/ml in 0.1M NaPO4 pH 7.5
(o-nitrophenyl-beta-D- galactopyranoside from Boehringer-Mannheim #810.088)

Extraction Buffer (50 ml)

		2.5 ml	        1M Tris  pH 7.8
		50  ul		1M DTT
		5.0 ml   	10% Triton X-100 
		42.45 ml 	diH20
  • Lyse the blood sample by mixing 2 ul fresh blood (RBC) sample with 300 ul of Extraction Buffer in a 1.5 ml tube. Include a positive control RBC sample from a LacZ-containing mouse as well as a negative control RBC sample from a wild-type C57BL/6 mouse.

  • Make up a Master Mix of the following components per each reaction: 

    	3 ul	100X Magnesium Chloride  Solution
       66 ul	1X ONPG Solution	
      201 ul 	0.1M Sodium Phosphate Solution

  • Take 10 ul of each RBC extract and add 20 ul of Extraction Buffer into a glass 12 x 75 mm borosilicate glass tube.

  • Add 270 ul of Master Mix to each reaction. Total volume will now be 300 ul. Let the reaction go for up to 90 minutes at room temperature, or until a dark yellow color change is noted. (You may have to let the reaction go overnight, but you must remember to cover the tubes to prevent any evaporation and to keep the reaction in the dark). This color change indicates that the beta-galactosidase has hydrolyzed the colorless ONPG substrate to its o-nitrophenol (yellow) and galactose products.

  • Set-up a control to subtract the background RBC contribution to the eventual color assessment. To 10 ul of RBC extract (from any blood sample), add 290 ul of Extraction Buffer and no Master Mix.

  • Set-up a blank with 30 ul Extraction Buffer + 270 ul Master Mix.

  • Stop all reactions with the addition of 500 ul of either diH20 or Sodium Carbonate solution.

  • Read on a spectrophotometer at OD 420 on a visual light setting using glass cuvettes. Linear measurements of the assay range from 0.0 - 1.0 OD. Zero the spectrophotometer with the blood background control.

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