The blots were incubated in the hybridization buffers indicated, following the manufacturer's recommendations for time and temperature. Six identical Northern blots were assayed for p53 using 106 cpm/ml of a random-primed, labeled probe. ULTRAhyb™ Detects a Signal in Only 8 Hours, Compared to a Minimum of 5 Days Using Competitors' Hybridization Solutions. 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.įigure 1.
Using ULTRAhyb with Northerns, mRNAs too rare to detect using standard means are detected in an overnight exposure. The benefits of greater sensitivity are remarkable. ULTRAhyb™, is as much as 100 times more sensitive than commonly used hybridization buffers (Figure 1). In many cases, maximizing the sensitivity of blot hybridization would forestall the need to switch to PCR-based detection methods, which are more difficult to set up and potentially less quantitative.Īmbion has developed a hybridization buffer that maximizes hybridization efficiency without increasing non-specific background. This limitation reduces the sensitivities of blot-associated assays by 20–100-fold, greatly reducing the ability to detect rare messages with Northern blots and dot blots, or to analyze limited DNA samples by Southern blotting. This is consistent with published work (1). Research at Ambion indicates that under typical blot hybridization conditions, only 0.5-5% of the target molecules on a membrane are actually bound by available probe. A primary limitation of all blot hybridizations is the efficiency of hybridization between the nucleic acids on the membrane, and the labeled nucleic acids in the hybridization solution.
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