12)After the hybridization wash the blot as follows:
a)rinse briefly in 2x SSC, 0. 1 % (w/v) SDS
b)twice, 5 minutes each in 2x SSC, 0. 1 % (w/v) SDS
c)twice, 10 minutes each in lx SSC, 0.1% (w/v) SDS
d)four times, 5 minutes each in 0.lx SSC, 0.1% (w/v) SDS
13)Remove the blot from the last stringency wash, drain and wrap in SaranWrap and expose to X-ray film, for example Hyperfilm MP. Keep the blot moist if it is to be reprobed.
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 elevated backgrounds following hybridization.. The Denhardt’s hybridization buffer may be stored at -20°C if required.
2) 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.
3) Hybond-XL has been designed for use with very low volumes of hybridization buffer (30-70µl/cm2). High backgrounds will result if sub optimum volumes are used for the membrane and hybridization conditions.
4) If there is significant overlap of the blot use of a nylon mesh should be considered. The mesh achieves separation of the blot layers allowing better probe access to these areas. It is strongly advised that hybridization volume should be increased (70-125µl/cm2). The nylon mesh should be at least 0.5cm larger than the blot. Place the mesh in the pre-wetting solution before the blot, in subsequent manipulations treat as "one". The nylon mesh may be reused after washing in 10% (w/v) SDS and extensive rinsing in distilled water.
5) It is important not to allow air to become trapped between the inner surface of the tube and the membrane. This can cause areas of no signal or background following hybridization.
6) Hybond-XL has been designed for use with low volumes of hybridization buffer (30-70µl/cm2).
7) High backgrounds will result if sub optimum volumes are used for the membrane and hybridization conditions.
8) 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.
9) Avoid placing the probe directly on the blot. Probe may be added to the hybridization while the tube is in a vertical position. If necessary probe may be mixed with a portion of the hybridization buffer and added to the tube in a larger volume.
10) 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 asTm-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
11)Washing in boxes is much more effective and is recommended if feasible. The inefficiencies of washing in tubes may be overcome by increasing the number of stringency washes while maintaining the same total wash time.
12)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.
