DIG Easy Hyb™ Granules Protocol & Troubleshooting

Product No. 11796895001

Protocol

Use of DEPC treated water

DEPC (diethylcarbonate) can be ordered from chemical suppliers (e.g., Sigma, Cat. Nr. D 5758). DEPC is used to produce RNase-free water. RNases are extremely stable enzymes capable of surviving autoclaving.

In the fumehood, add 1ml of DEPC to 1 liter of deionized, distilled water while stirring. Stir for at least 2 hours. Autoclave DEPC-water for 20-30 minutes to evaporate residual DEPC. After cooling to room temperature (+15 to +25 °C) the water is ready to use. For RNA in situ hybridization, Roche recommends using DEPC treated water t o prepare all aqueous solutions.

Note: For best results in the protection of mRNA in in vitro transcription reactions, use Roche Protector RNAse Inhibitor.

Reconstitution in DEPC water

DIG Easy Hyb Granules are reconstituted by carefully adding 64 ml sterile, redist. water in two portions to the plastic bottle and are dissolved by stirring immediately for 5 min at +37 °C. For applications with northern blots, DIG Easy Hyb Granules must be dissolved under RNase-free conditions using DEPC-treated water. The volume of 64 ml fits exactly in the plastic bottle; for convenience, divide the 64 ml into two portions. Granules can be dissolved over night at RT. For best results, do not autoclave DIG Easy Hyb Granules in solution.

Convenient ready-to-use DIG Easy Hyb solutions are also available from Roche.

Troubleshooting

Additives do not enhance hybridization sensitivity

For [32P]-labeled probes, it has been reported that additives to the hybridization buffer can increase hybridization sensitivity. For example, adding 10% dextran sulfate or 6– 10% PEG 8000 can increase the hybridization sensitivity two- to threefold.

However, Roche testing has shown that adding dextran sulfate or PEG to DIG Easy Hyb Bbuffer does not increase the sensitivity of DIG-labeled probes. Roche does not recommend using dextran sulfate and PEG 8000 with the DIG System, due to the significant lot-to-lot variation of both products. In general, for best results, do not add supplements to DIG Easy Hyb Buffer; use it as supplied for nonradioactive hybridization experiments.

Materials

     

DIG EASY HYB is a trademark of Roche

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