1. Probe Preparation
*For simultaneous hybridization of two CEP probes, substitute 7 µL CEP hybridization buffer, 1 µL purified H2O, and 1 µL each of two different CEP DNA probes.
2. Slide Preparation
Fluorophores are readily photo-bleached by exposure to light. To limit this degradation, handle all solutions containing fluorophores in reduced light, including those steps involved in handling the hybridized slide.Perform all steps which do not require light for manipulation (incubation periods, washes, etc.) in darkness.
The use of a calibrated thermometer is highly recommended for measuring the temperature of solutions.
Prior to first opening each vial of reagent, centrifuge for 2-3 seconds using a standard bench-top microcentrifuge.
Place room temperature Coplin jar containing the denaturant solution in a 73+/-1°C water bath approximately 30 minutes prior to use.
If more than four slides have been hybridized they must be washed in more than one batch. The temperature of the wash solution must return to 73+/-1°C before washing each batch.
20X SSC (for 500 mL of 20X SSC, pH 5.3)
Add 132g 20X SSC to 400 mL H2O and mix thoroughly. Adjust pH at room temperature with a pH meter to 5.3 using concentrated HCl and adjust to final volume of 500 mL. Filter through a 0.45 micron pore filtration unit. Store up to six months at room temperature.
2X SSC/0.1% NP-40 (for 1 liter)
Add 100 mL 20X SSC (pH 5.3) to 850 mL purified H2O. Add 1.0 mL NP-40. Adjust pH to 7.0-7.5 with NaOH using a pH meter. Add H2O to bring final volume of the solution to 1 liter. Store up to six months at room temperature.
0.4X SSC/0.37 NP-40 Wash Solution
Add H2O to bring final volume of the solution to 1 liter. Filter through 0.45um pore filtration unit. Store up to six months at room temperature. Note: The wash solution must be at 73+/-1°C before using. In order to maintain the proper temperature range, four slides MUST be placed in the heated solution at one time. If fewer than four slides have been hybridized, room temperature microscope slides must be used to bring the number to four slides. If more than four slides have been hybridized they must be washed in more than one batch. The temperature of the wash solution must return to 73+/-1°C before washing each batch. Do not remove the coverslips from several slides before placing any of the slides in the wash bath. Begin timing the incubation when the last slide has been added to the wash bath.
Denaturing Solution (for 70 mL)
Add 49 mL formamide, 7 mL 20X SSC (pH 5.3) and 14 mL purified H2Oto a glass Coplin jar and mix thoroughly. Measure pH at room temperature using pH meter to verify pH is between 7.0-8.0. Use each batch of denaturant for seven days and then discard. Between periods of use, store at 4°C.
Ethanol Wash Solutions
For final concentrations of 70%, 85% and 100%. Prepare v/v dilutions of 100% ethanol with H2O.Use dilution for up to seven days and then discard. If solution evaporates or becomes diluted, replace with fresh solution. Between periods of use, store at room temperature.
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