Blotting and Hybridization of a probe generated by the NEBlot Phototope Kit.

Protocol

1. Standard Hybridization
   
   Perform gel electrophoresis using standard techniques (3,4,5).  Run 1 μl of biotinylated markers on the gel.  If the target DNA/RNA concentration is low, dilute the markers to reduce their intensity.
   
   
2. Wash Step - Detection Solution A
   
   Add to the hybridization bag, 0.1 ml of Detection Solution A per cm² of membrane. 
   
   Incubate for 5 minutes at room temperature with moderate shaking. 
   
   Drain the solution before the next step.
   
   Sample calculation
   100 cm² x 0.1 ml/cm² = 10 ml
   
   
3. Transfer the target DNA/RNA to a nylon membrane (See Appendix III of product manual) using standard techniques (3,4,5).  Covalently bind the DNA/RNA to the membrane by UV cross-linking or other standard techniques.
   
   
4. Place the membrane in a hybridization bag or container and prehybridize in your chosen hybridization buffer, using standard protocols (3,4,5), for 1 hour.  The membrane should be completely covered by the prehybridization solution.
   
   
5. Denature the biotinylated probe by placing in boiling water for 5 minutes, chilling on ice for 5 minutes, and centrifuging briefly to collect at the bottom of the tube.  Add to the prehybridization buffer in the bag or the container.
   
   
6. Hybridize the probes to the nucleic acids immobilized on the membrane using standard protocols (3,4,5,).
   
   
7. Storing the membrane
   
   The membrane can be stored in a small volume of 1X SSC in the sealed bag at room termperature for the short term.  For extended storage, freeze at -20^oC.  Do NOT allow the membrane to dry out.
   
   
8. Storing the probe.
   The probe, in hybridization buffer, can be stored at -20^oC for subsequent re-use.
   Note:  The probe must be denatures by boiling for 5 minutes and chilling on ice before re-use.
   
   
9. Stripping and Reprobing
   After detection with one probe is complete, the membrane can be stripped and washed to remove the probe.  Hybridization with another probe can then be performed beginning with the prehybridization step.
   
   
10. Stripping Southerns
    Rinse the membrane in water.
    
    
11. Incubate in 0.4 NaOH, 0.1% SDS at 80^oC for 30 minutes.
    
    
12. The membrane can now be reprobed.
    
    
13. Stripping Northerns
    Rinse the membrane in water.
    
    
14. Incubate in a large volume (~ 500 ml) of 2mM EDTA, 0.1% SDS at 80^oC for 15 minutes.
    
    
15. Rinse membrane in 2X SSC at room temperature.
    
    
16. The membrane can now be reprobed.
    
    Note: Incubating for more than half an hour (overstripping) may adversely affect the target RNA/DNA bound to the membrane.