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Download: MSDS PDF

Description:
The NEBlot® Phototope® Kit is designed for the chemiluminescent detection of nucleic acids in standard Southern and Northern blotting applications. With the NEBlot Phototope Kit, biotin is incorporated into hybridization probes in two ways- first, by utilizing a unique biotinylated random octamer primer and second, by incorporating biotinylated dATP during polymerization. The biotinylated probes are hybridized to target DNA immobilized on a membrane, and the target DNA is detected by chemiluminescence using one of the Phototope Detection Kits (sold separately).

Advantages:

• Stability: Biotinylated probes and reagents are stable for extended periods unlike ³²P-labeled probes which are only stable for a few days.
• Speed: Only 40 minutes is required for the entire detection procedure. Exposure times of 5-10 minutes.
• Multiple Exposures: Light is emitted at a constant rate for days, so you can perform multiple exposures to optimize signal intensity. Re-exposure at a future date is achieved by simply adding more chemiluminescent reagent.
• Simple Reprobing: If you want to search the same membrane for a different set of fragments, simply strip the membrane and rehybridize with a different probe.
• Versatility: The Kit can be used for Northern blotting, Southern blotting, plaque or colony hybridizations.
• Simultaneous Detection of Biotinylated Molecular Weight Standards
• No Radioactivity

Kit Components:
5X Labeling Mix
Biotinylated dNTP Mixture
Klenow Fragment (3´→5´ exo–)
Pre-biotinylated Molecular Weight Markers
Unbiotinylated Control DNA

Storage Conditions

Storage Temperature:
-20°C

Notes

General notes:

1. Phototope® Kits require transfer of DNA from a gel to a solid support membrane. The kits were developed using Millipore Immobilon-S membranes; however, other nylon based membranes with a neutral or slight positive charge can be substituted. Nitrocellulose does not give acceptable results.  

FAQs

1. After labeling a probe with the NEBlot Phototpe Kit, I am seeing a weak signal;  How can I determine if the labeling of the probe is the problem?
2. How can I improve the priming reaction in the NEBlot Phototope Kit?
3. After labeling a probe with the NEBlot Phototope Kit, I am seeing a weak signal.  Other than labeling the probe, what else could be the problem?
4. Can biotinylated probes be used in gel-shift experiments?
5. Is it possible to detect a target RNA or DNA with a biotinylated probe that has been labeled with a single biotin?
6. How can I determine if the detection procedure is causing a high or uneven background with the NEBlot Phototope Kit?
7. What are other sources of high or uneven background with the NEBlot Phototope Kit?
8. Can you recommend a hybridization or detection bag?
9. Can I store the NEBlot Phototope Kit at minus 20^oC?
10. What is the difference between the NEBlot kit and the NEBlot Phototope kit?
11. How sensitive are the NEBlot® Phototope® Kits?
12. What are the advantages of using a chemiluminescence based detection system versus a radioactive method of detection?
13. What is the % of biotinylated dATP in the dNTP mixture provided with the NEBlot® Phototope® Kit?
14. What is the rate of biotinylated dATP incorporation in the probed synthesized using the NEBlot® Phototope® Kit?
15. Why isn’t the % of biotinylated dATP in the dNTP mxiture higher than 16%?
16. Where in the dATP molecule is the biotin attached?
17. Are biotinylated dNTPs sold separately?
18. How can the biotinylated probe be denatured?
19. When I run my probe in a gel, I see a smear. Why?
20. What probe size distribution can I expect to obtain by synthesizing a biotinylated probe with the NEBlot Phototope kit?
21. Can I produce biotinylated probes by using DNA templates that are shorter than 100 bps?
22. Should the unincorporated nucleotides be removed from the biotinylated probe mixture prior to their use for detection?
23. Do you recommend the QIAquick PCR purification kit to remove the unincorporated nucleotides?

Protocols

Protocols for NEBlot® Phototope® Kit

Southern Blot using the NEBlot Phototope Kit. Exposure time 10 minutes.

References

1. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 132, 6-13.
2. Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem., 137, 266-267.
3. Denhardt, D.T. (1966) Biochem. Biophys. Res. Commun., 23, 641.
4. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.), 9.31-9.62.
5. Perry-O'Keefe, H. and Kissinger, C.M. (1989) F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology, pp. 3.19-3.19.8.
   

Reagents Sold Separately

5X Labeling Mix
Biotinylated dNTP Mixture
Klenow Fragment (3´→5´ exo–)

Companion Products

Phototope®-Star Detection Kit