Tools for Blot Hybridization
|A mainstay of molecular biology research is the nucleic acid blot. "Blots" are membranes such as nitrocellulose or coated nylon to which nucleic acids have been permanently bound. Blot hybridizations with specific nucleic acid probes provide critical information regarding gene expression and genome structure. The most common blot applications used in modern laboratories are Northern blots, Southern blots and dot/slot blots. Regardless of the type of blot, the principles of probe synthesis, hybridization, washing and detection are the same. Ambion has invested substantial research efforts into understanding and overcoming the limitations of each of the steps common to all blot analyses. The following article outlines some of these advancements and the products that have resulted from our efforts.|
Maximizing Blot Sensitivity
Ambion has developed a hybridization buffer that maximizes hybridization efficiency without increasing non-specific background. Our ultrasensitive hybridization buffer, ULTRAhyb™, is as much as 100 times more sensitive than commonly used hybridization buffers (Figure 1). The benefits of greater sensitivity are remarkable. Using ULTRAhyb with Northerns, mRNAs too rare to detect using standard means are detected in an overnight exposure. Single copy genes are readily detected in only 1 µg of genomic DNA by Southerns only compared to the 10 µg typically required when using standard hybridization buffers.
Figure 1. ULTRAhyb™ Detects a Signal in Only 8 Hours, Compared to a Minimum of 5 Days Using Competitors' Hybridization Solutions. Six identical Northern blots were assayed for p53 using 106 cpm/ml of a random-primed, labeled probe. The blots were incubated in the hybridization buffers indicated, following the manufacturer's recommendations for time and temperature. All blots were washed identically using 2X SSC/0.1% SDS and 0.1X SSC/0.1% SDS. The blots were exposed to the same piece of film at -80°C with one intensifying screen for the indicated times.
1. Molecular Cloning: A Laboratory Manual, 1989. Sambrook, J. Fritsch, E.F., Maniatis, T. Cold Spring Harbor Laboratory Press.
2. Current Protocols in Molecular Biology, 1994. Ausubel, F.M. and others, editors. John Wiley & Sons, Inc.
QuikHyb, Rapid-hyb and ExpressHyb are registered trademarks of Stratagene, Amersham and CLONTECH, respectively.
Reagents for Northern and Southern Blotting
RNase-free Wash Buffers
Compatible Reagents for Blot Hybridization
- Vernier, P., Mastrippolito, R., Helin, C., Bendali, M., Mallet, J., Tricoire, H. (1996) Radioimager quantification of oligonucleotide hybridization with DNA immobilized on transfer membrane: application to the identification of related sequences. Analytical Biochemistry 235, 11-19.