1.2.1. Slide Denaturation

1. Denaturation buffer(preferred): 70% (v/v) deionized formamide, 10% (v/v) filtered double-distilled water, 10% (v/v) 20X SSC, 10% (v/v) phosphate buffer; make fresh as required.

OR:   Denature solution: 70% (v/v) formamide, 2X SSC (pH7.0), 0.1mM EDTA, pH7.0
Add 175mL formamide, 25mL 20X SSC (pH7.0), 50mL 0.5M EDTA, pH7.0 and 50mL purified H₂O to make 250mL solution and mix thoroughly.  Verify that the pH is 7.0-7.5 by measuring the pH at ambient temperature.  Between use, store covered at 4℃.  Discard after 7 days.
2. Deionized formamide: Add 5 g of ion exchanger Amberlite MB1 (Serva) to 100 mL of formamide (Merck, Darmstadt, Mannheim, Germany) stir for 2 h (room temperature) and filter twice through Whatmann no. 1 filter paper. Aliquot and store at -20ºC.
3. Phosphate buffer: prepare 0.5M Na₂HPO₄ and 0.5M NaH₂PO₄, mix these two solutions (1: 1) to get pH 7.0, then aliquot and store at -20ºC.

1.2.2. Probe Denaturation

1.   Hybridization buffer: Dissolve 2 g dextran sulfate in 10 mL 50% deionized formamide/2X SSC/50 mM phosphate buffer for 3 h at 70ºC. Aliquot and store at -20ºC.

OR: Hybridization solution: MM2.1:    5.5mL formamide
                                                      1g Dextran sulfate
                                                       0.5mL 20X SSC

Heat to 70℃ for several hours to dissolve the dextran sulfate, then cool and adjust to pH 7.0 and add water to volume of 7mL.

1.2.3. Posthybridization and Detection Washing

1. Washing solution I: 50% (v/v) formamide (Merck), 10% (v/v) 20X SSC, 40% (v/v) distilled water; make fresh as required.
2. Washing solution 2 (WS-2)(4X SSC/0.05% Tween 20).
3. Washing solution 3 (WS-3)(4X SSC).
4. Blocking solution: 1-3% (w/v) BSA in 4X SSC, 0.05% (v/v) Triton X-100 (make up fresh).
5. PN buffer: 0.1M NaH₂PO₄/0.1M Na₂HPO₄ M, pH 8.0; 0.1% NP-40.
6. PNM buffer:
Add 5% (w/v) non fat dry milk to PN buffer plus 0.02% (w/v) Na-azide, incubate at 37℃ overnight. It will look terrible. Centrifuge the solution for 5 minutes at 1000g. Transfer the supernatant to a clean tube and store at 4℃.
7. FITC-Avidin (2mg/2mL): Add 398mL PNM buffer to make up 5mg/mL.

OR: Solution 1: FITC-avidin (CAMON Vector Laboratories)/4X SSC/0.2 %Tween/5% BSA (1: 300 both Sigma, St. Louis, MO); make fresh as required.
8. Anti-Avidin (2mg/2mL): Add 398mL PNM buffer to make up 5mg/mL.

OR: Solution ll: Biotinylated antiavidin (CAMON Vector Laboratories)/Anti-digoxigenin- rhodan-dne (Boehringer Mannheim, Germany)/4X SSC/0.2%Tween/5% BSA (1: 20: 100); make fresh as required.

上一页  [1] [2] [3] [4] [5] [6] 下一页