8. DEPC treated H20 (2.1.3.5).
9. 0.2 M Tris pH 8 (Dissolve 121.1 gms Tris-base in water, adjust pH with concentrated HCl; pH of Tris is temperature sensitive, sterilize by autoclaving).
10. 10X DNaseI Buffer (0.2 M Tris pH 8, 0.1 M MgCl₂.
11. DNaseI mix (1U DNaseI (Amersham Pharmacia Biotech) (RNase-free, store at -20℃), 20 mM Tris pH 8, 10 mM MgCl₂,, 10mm DTT).
12. 1 M Dithiothreitol (DTT) (Sigma) (Dissolve 3.09 g of DTT in 20 ml of 0.01 M sodium acetate pH 5.2, sterilize by filtration, aliquot and store at -20℃).
13. 0.2M Na₂CO₃ pH 10.2
14. 7.5M NH₄OAC.
15. Absolute ethanol.
16. Resuspension buffer (50% formamide, 5mM Tris-HCl pH 7.5, 0.5mM EDTA and 0.01% Tween 20).
17. Formamide (Sigma).
18. TE pH 7.5 (10 mM Tris-HCl pH 7.5, 1 mM EDTA).
19. Tween 20 (polyoxyethykenesorbitan monolaurate) (Sigma).
20. Aluminum plate sealers or 8-strip PCR caps (E&K Scientific).

1.1.5. RNA Probe Quantificaton
1. DIG Quantification Teststrips (Roche).
2. DIG Control Teststrips (Roche).
3. 300 µl/well 96-well plate (E&K Scientific).
4. Multichannel pipette (Brand).
5. Falcon Pipetaid.
6. Aluminum plate sealers or 8-strip PCR caps (E&K Scientific).
7. Positively charged Nylon membrane (Roche).
8. UV Stratlinker (Stratagene).
9. HybAid tubes (HybAid Limited).
10. HybAid Hybridization oven (HybAid Limited).
11. 1X blocking solution (Dissolve 10 g of blocking reagent (Roche) in 0.1M maleic acid, 0.15M NaCl adjust pH to 7.5 with NaOH (solid), store at 4 ° C).
12. Tween 20 (polyoxyethykenesorbitan monolaurate, Sigma).
13. Anti-Digoxigenin-AP Fab Fragments (Roche).
14. AP Buffer (0.1M NaCl, 0.05M MgCl₂, 0.1M Tris pH 9.5, 0.1% Tween 20; prepare fresh before use).
15. Developing solution (Roche, 45 µl NBT and 35 µl BCIP, store at -20℃) per 10 ml of AP Buffer; add NBT/BCIP just before use).

1.2. 96- well Plate RNA in situ Hybridization
1.2.1. Embryo Collection
1. Foam Trays (Xpedex).
2. Fly Food¹ (3500 ml H₂O, 125 g Agar, 125 g Sucrose, 8 g p-Hydroxybenzoic Acid Methyl Ester, 1360 ml Grape Juice, 100 ml 1.25 N NaOH).
3. Yeast (Fleishman) - Mix yeast with H₂O to form a thick paste.

1.2.2. Embryo Fixation
1. Three-level sieve (nominal sieve openings (850 µm (20), 250 µm (60), 150µm (100)) (Fisher).
2. Falcon Pipetaid.
3. Paint brush (Winsor & Newton).
4. Crystallization dish (Fisher).
5. 100% sodium hypochlorite (commercial bleach, Kem Tek).
6. Gyrotory¨ shaker Model G2 (New Brunswick).
7. Heptane (Fisher).
8. Formaldehyde (Sigma, ACS reagent grade, 25 ml aliquots).
9. DEPC treated H₂O (2.1.3.5).
10. PBS (Dissolve 8 g of NaCl, 0.2 g KCl, 0.24 g KH₂PO₄ and 2.72 g Na₂HPO₄ ·7H₂O in 800 ml of DEPC treated H₂O, adjust pH to 7.4 with HCl, adjust volume to 1L, sterilize by autoclaving).
11. Methanol (EM Science).

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