Molecular Research Center Washing & Pre-Hyb Solution

WASHING & PRE-HYB SOLUTION

Cat. No. WP 117
Store at room temperature.
The product is stable for at least one year after the date of purchase.

PRODUCT DESCRIPTION
Washing and Pre-Hyb solution is supplied as a 10X concentrate for use in northern and Southern blotting. This solution allows both pre- and post-hybridization washes to be performed at room or elevated temperatures. It can be used with a variety of charged or non-charged nylon and nitrocellulose membranes and with radioactive or non-radioactive detection systems. The Washing & Pre-Hyb Solution is recommended for use with the High Efficiency Hybridization System (cat. nos. HS 114 and HS114F) and is a component of the Super Hyb ^TM Kit (cat. nos. SK 116 and SK 116F).

APPLICATION NOTES
Dilute an aliquot of the 10X Washing & Pre-Hyb Solution to obtain a 1X working solution. The 1X working solution is stable for two weeks after dilution. Use the 1X working solution for pre- and post-hybridization washes and for removal of the probe from the membrane. In addition to the information presented here, see the High Efficiency Hybridization System protocol for more information.

PREHYBRIDIZATION WASH
Place a hybridization membrane in a hybridization bag, tray or rotating tube and add a sufficient amount of the 1X Washing & Pre-Hyb Solution to cover the membrane. Perform prehybridization for at least 20 minutes in a rotating tube or by leaving the tray or bag in a horizontal position at room temperature. Supplementation of the 1X prehybridization solution with salmon sperm DNA or RNA is not necessary. Remove the prehybridization solution and proceed with hybridization steps according to preferred protocol.

POSTHYBRIDIZATION WASHES
Following hybridization, place the membrane in a tray with 1X Washing & Pre-Hyb Solution. The membrane should float freely in the washing solution. Place the tray on a shaker and shake the membrane for 7-10 minutes at room temperature. Pour off the washing solution. Repeat the washing cycle five times at room temperature. After washing, wrap the wet membrane in plastic wrap (Saran/Glad) for autoradiography. Alternatively, proceed with blocking and binding steps specific to non-radioactive detection procedures. Keeping the membrane wet during detection procedures is critical for probe removal before the next hybridization cycle.

A standard washing protocol for hybridization membranes includes room temperature washes followed by washes at 50 - 60 C. The wash at elevated temperature diminishes binding of a hybridization probe to the membrane and dissociates hybrids formed between the probe and non-target RNA or DNA sequences. We have found that when using the High Efficiency Hybridization System for hybridization, the membrane wash at elevated temperature is not necessary. These hybridization solutions effectively reduce binding of the probe to the hybridization membrane and minimize mismatches of the probe with non-target RNA or DNA sequences. Due to this blocking of non-specific background, all washes may be preformed at room temperature. Room temperature washes are advantageous when used in conjunction with probes prepared with either an insert cDNA or the whole vector containing the insert and plasmid DNA.

NOTE: When using hybridization systems other than Molecular Research Center's High Efficiency Hybridization System or probes yielding high background, perform the last two post hybridization washes at 50 C in a shaking water bath or in the hybridization oven with rolling tubes. Shaking (or rolling) is critical for the efficient removal of a probe from non-specific binding sites on a membrane.

PROBE REMOVAL FROM NYLON MEMBRANES
Place the wet membrane in a hybridization bag filled with 1X Washing & Pre-Hyb Solution. Place the bag in boiling water for 5 minutes. Remove the 1X working solution and repeat this washing process 3 - 4 times.

REFERENCES

Cornish EC, Beckham SA and Maddox JF (1998) Southern hybridization revisited. Biotechniques 25, 947-953.