DAY 2 SOLUTIONS
| PROTEINASE K STOCK. Stock is liquid proteinase K from Boehringer Mannheim, catalog # 13330224-04. The stock concentration varies from batch to batch (10-15 mg/ml). Concentrated stock is stored at 4℃. Just prior to use, stock is diluted to 200 ug/ml using 20 mM Tris-HCl pH 7.8, 2mM CaCl2. | RNASE STOCK SOLUTION. 10 mg/ml RNase A is made DNAse free as described in Maniatis. Stock is stored in 1 ml aliquots at -20℃ |
| FORMAMIDE (triple distilled): ONCOR formamide (Catalog # S4117) ONCOR, 209 Perry Parkway, Box 870, Gaithersburg, Maryland 20877 |
DENATURING SOLUTION Final conc. Recipe for 60 ml 70% Formamide 42 ml Formamide 2X SSC 6 ml 20X SSC 12 ml H2O |
| 20 X SSC Final conc. Recipe for 1 liter 3.0 M NaCl 175.3 g NaCl 0.3 M Na Citrate 88.2 g 800 ml H2O pH to 7.0 using NaOH then bring to 1000 ml using H2O |
10 X SSCP Final conc. Recipe for 100 ml 1.5 M NaCl 8.75 g NaCl 0.15M Na Citrate 4.41 g 0.2 M NaH2PO4 2.4 g NaH2PO4 pH to 6.0 using NaOH |
| HYBRIDIZATION SOLUTION Final conc. Recipe for 10.4 ul probe DNA 1ul probe DNA (approp. conc.) 50% Formamide 5 ul Formamide 10% Dex, Sulfate 2 ul 50% Dextran Sulfate 2X SSCP 2 ul 10X SSCP 400 ug/ml salmon 0.4 ul 10 mg/ml sonicated sperm DNA salmon sperm DNA |
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DAY 3 SOLUTIONS
| POST-WASH SOLUTION Final conc. Recipe for 60 ml 60% Formamide 36 ml Formamide 2X SSC 6 ml 20X SSC 18 ml H2O |
1X PBD 0.1 M NaH2PO4 0.1 M Na2HPO4 0.1% (w/v) Nonadet P40 |
| ANTIFADE/PROPIDIUM IODIDE Dissolve 50 mg p-phenylenediamine (Sigma) in 5 ml 1X PBS, adjust to pH 9.0 Add 45 ml glycerol & stir to homogeneity. We store this in 1 ml aliquots at -80 in the dark. When using for FISH we add 5µl of propidium iodide (2.5mg/ ml stock solution in H2O) to 1 ml and keep this dark at -20. |
SLIDES. 10 well slides with 5 mm diameter circular wells (autoclavable) obtained from Roboz Surgical Instrument Co. Inc., 9210 Corporate Blvd., Suite 220, Rockville, Maryland 20850). |
