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.,  Cat. No. D5758). 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 to 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 overnight 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™ buffer 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.