Prehybridization And Hybridization

1. Prepare hybridization buffer based on nonradioactively or radioactively labeled DNA. One filter (10 x 12 cm2) needs 10 to 15 ml buffer, or 30 to 40 ml of buffer is sufficient for four to six filters in a single hybridization container.

2. If the blotted membrane is predried, immerse it in 5X SSC for 5 min at room temperature to equilibrate the filter.

Note: Do not allow the filters to dry out during subsequent steps. Otherwise, a high background or anomalous results will occur.

3. Carefully roll the filter and insert it into a hybridization bottle or an equivalent container containing 10 to 15 ml/filter of appropriate prehy-bridization solution, depending on the isotopic or nonisotopic DNA probe used. Cap the bottle or container and place it in a hybridization oven or equivalent shaker with controlled speed and temperature. Allow prehy-bridization to proceed for 1.5 h with gentle agitation.

Notes: (a) It is strongly recommended not to use traditional plastic hybridization bags because it is usually not easy to get rid of air bubbles and they cannot be well sealed. This will result in leaking and contamination. An appropriate size of plastic beaker or tray or hybridization bottle is the best type of hybridization container to use for this purpose. (b) The temperature depends on the prehybridization buffer; it should be 42 to 55°C if the buffer contains 30 to 40% (for low-stringency conditions) or 50% (for high-stringency conditions) formamide. If the buffer contains no for-mamide, the temperature can be 65°C. High-stringency conditions can prevent nonspecific cross-binding during hybridization. Assuming that the buffer used for this experiment contains 50% formamide, in our experience, the temperature for this experiment can be set up at 42°C regardless of which DNA labeling method in step 3 is employed. (c) It is highly recommended that a regular culture shaker with a cover, controlled speed, and temperature be used as a hybridization chamber. Such a chamber is easy to handle by simply placing the hybridization beaker or tray containing the filters and buffer on the shaker in the chamber. Multiple filters can be placed in one container containing an appropriate volume of hybridization buffer. This is particularly useful for biotech lab classes. In contrast, a commercial hybridization oven may be difficult to operate. Rolling up the filters with a matrix screen and placing the roll inside the hybridization bottle takes time and air bubbles are easily generated during that period.

4. If the probes are dsDNA, denature the probe to ssDNA in boiling water for 10 min and immediately chill on ice for 4 min. Briefly spin down.

Note: This is a critical step. If the probes are not completely denatured, a weak, or no hybridization signal will occur. However, the probes prepared by Method A do not need denaturation.

5. Dilute the probes with 0.8 ml of hybridization solution. Replace the prehybridization buffer with fresh prehybridization buffer and add the diluted probes to the freshly replaced prehybridization buffer. Alternatively, diluted probes may be directly added to the old prehybridization buffer without replacing it. Place the container back in the hybridization oven or shaker and allow hybridization to proceed for 6 h to overnight under the same conditions as prehybridization.

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