Protocol
1)Prepare the hybridization buffer, for example
Denhardt’s Buffer
Modified Church Buffer
5x SSC
0.5M phosphate buffer, pH 7.2
5x Denhardt's solution
7% (w/v) SDS
0.5% (w/v) SDS
l 0mM EDTA
Ensure the SDS is in solution before use. Gentle
heating may be necessary
Combine all the components, make up to the required volume.
2) Prepare the radiolabelled probe using the appropriate procedure.
3) Preheat the required volume of hybridization buffer to an appropriate temperature.
4)Pre-wet the blot in a suitable dish, first in water then in an appropriate buffer. Ensure that the nucleic acid side is uppermost. Roll the blot along its length in such a way as to minimise overlap in the tube. Place inside the hybridization tube.
5)Add a small volume of appropriate buffer to the hybridization tube, cap the tube. Unroll the blot by rotating the tube in the opposite direction to the "rolled" blot.
6)Drain the tube of excess liquid and replace with the appropriate volume of hybridization buffer.
7) Prehybridize for 30 minutes at the appropriate temperature. Ensure that the tube is placed in the correct orientation within the oven to avoid "rolling' up of the blot.
8) When using labelled double stranded probes, pipette the required amount into a clean microcentrifuge tube. If the volume is less than 2ml, make up to this volume with water or TE buffer. Denature the probe by boiling for 5 minutes and snap cool on ice. Briefly centrifuge to draw the contents to the bottom of the tube
11) Prepare the stringency wash solutions. The wash solution should be used in excess. Use a volume that occupies 33-50% of the tube.
Low stringency wash;
2x SSC, 0.1% (w/v) SDS
Medium stringency wash:
lx SSC, 0.1% (w/v) SDS
High stringency wash:
0.lx SSC, 0.1% (w/v) SDS
