CGlobal Precautions

• Physically separate reagents and materials for in situ hybridization from those used for other procedures, including other RNA methods.

• Clean glassware with a neutral detergent, rinse thoroughly, and bake at 240-260°C for >4 h.

• Whenever possible, use RNase-free, DNase-free, disposable plasticware, and sterilize solutions by filtration (0.22 |im) to avoid autoclaves, a potential source of RNase contamination.

• Treat water used for making solutions with diethyl pyrocarbonate, an RNAase inhibitor (DEPC-H2O). Use very high-quality water at all steps.

• Be rigorous in cleanliness. Wear gloves and change them frequently. Cover work surfaces with clean bench paper. Keep work areas and equipment clean and dust-free.

These precautions are essential to simplify the process of troubleshooting failed experiments.

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