Generation of a Labeled Probe using the NEBlot Phototope Kit
- 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.
- Denature in boiling water for 5 minutes.
- Quickly place on ice for 5 minutes.
- Centrifuge briefly at 4oC.
- 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-)
- Incubate at 37oC for the calculated reaction time. (See Appendix II of the product manual)
- Terminate the reaction by adding 5 μl of 0.2 M EDTA, pH 8.0.
- Separate the synthesized probes from unincorporated nucleotides by gel filtration, spin column, or ethanol precipitation (3,4,5)
- Resuspend in 20 μl of 1X TE.
- 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 -20oC