Generation of a Labeled Probe using the NEBlot Phototope Kit

Overview

Protocol

1. Dilute 10 ng-500 ng of template DNA (1-34 μl) in nuclease free H₂O to a total volume of 34 μl.
   In a separate tube, add 2 μl (1 μg) of unbiotinylated control DNA to 32 μl nuclease free H₂O.
2. Denature in boiling water for 5 minutes.
3. Quickly place on ice for 5 minutes.
4. Centrifuge briefly at 4^oC.
5. Add the following reagents to the template and control tubes in the order listed.
   
   • 10 μl of 5X labeling mix (contains biotinylated random octamers)
   • 5 μl of dNTP mix (contains dNTPs and biotin-dATP)
   • 1 μl of Klenow Fragment (3’ → 5’ exo^-)
   
   
6. Incubate at 37^oC for the calculated reaction time. (See Appendix II of the product manual)
7. Terminate the reaction by adding 5 μl of 0.2 M EDTA, pH 8.0.
8. Separate the synthesized probes from unincorporated nucleotides by gel filtration, spin column, or ethanol precipitation (3,4,5)
9. Resuspend in 20 μl of 1X TE.
10. Determine approximate concentration of biotinylated DNA.
    
    Note:  A reaction using 100 ng of template in a 1 hour reaction will have resulted in the generation of approximately 300 ng of biotinylated DNA.  Therefore, the concentration, after resuspension in 20 μl, will be about 15-20 ng/μl.
    
    For long term storage, keep the resuspended probe at -20^oC