Notes
1)There are a wide variety of hybridization buffers used by researchers. This Denhardt's based buffer is that used in the quality control of all Hybond nylon membranes. A reduced concentration of SDS has been found to give elevated backgrounds following hybridization. The Denhardt’s hybridization buffer may be stored at -20°C if required. This modification of the Church and Gilbert buffer is routinely used at Amersham Pharmacia Biotech. It has been shown to be suitable for Southerns, Northerns, dot blots and library screening applications. The hybridization buffer may be stored at room temperature. Ensure the SDS is fully dissolved before use. This may be achieved with gentle heating.
2) For radioactive applications use a probe concentration of 0.5 - 2 x 106 incorporated counts per ml of hybridization buffer for single copy gene detection, (i.e. high sensitivity application) or 0.125 - 0.5 x 106 incorporated counts per ml of hybridization buffer for high target work, for example colonies/plaques, PCR products etc. Probe purification, to remove unincorporated radioactive nucleotides, is strongly recommended.
3) Pre-wetting in a suitable buffer is essential for large blots (>100cm2) or multiple blots. See Critical Parameters. Hybridization may be carried out in bags, or boxes, provided there is sufficient buffer for the container. Adequate circulation of the buffer is essential. When hybridising several blots together, the blot should move freely within the buffer.
4) Avoid placing the probe directly on the blots, as this will cause excessive background.
5) Hybridization temperatures may vary with the probe. Lower temperatures achieve lower stringency. The temperature of hybridization used will depend on the degree of homology between the probe and the target. 65-68°C is suitable for most long probes (>100bases). With short/oligo probes (<50 bases) hybridization temperature is usually defined as Tm-5°C: Tm (melting temperature) = (4x number of G+C bases) + (2x number of A+T bases) Hybridization time can also vary. Short hybridization times may be suitable for high target applications.
6) Stringency washes will depend on the nature of the probe and target to be hybridized. Salt concentration and temperature should be taken into consideration. The lower the salt concentration, the greater the stringency. The higher the washing temperature, the greater the stringency. Most commonly, stringency washes proceed from "high salt"/"low temperature", for example 5x SSC, 0.1% SDS at room temperature, to "low salt/high temperature", for example 0.lx SSC, 0. 1 % at 65°C (nominal hybridization temperature).
7) Some procedures include room temperature washes under low stringency conditions. Do not allow the SDS to come out of solution during these washes, significant levels of background may result. Adequate circulation of the stringency buffer is essential when washing. Washing in boxes is advised.
8) The use of SaranWrap with 35S labelled probes will significantly increase exposure times. In this case the blot should be air dried before autoradiography, if reprobing is not required.
Hybridization in tubes
There are numerous commercially available rotisserie devices suitable for use as hybridization ovens. These can accommodate 2 to 24 hybridization tubes. The major advantage of this approach to hybridization is the use of low volumes of hybridization buffer, and therefore minimal probe volumes. This is achieved because fluid is able to move continually over the membrane.
