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The Ambion® kit is for labeling DNA using a random-priming method, which has technical improvements over existing methods including the use of a high-purity, exonuclease-free Klenow and Random Decamers to produce probes with greater than 109 cpm/µg in 10 min reactions. The kit includes sufficient reagents for 30 reactions.
• Fast, 10 min reaction time • Elimination of Klenow exonuclease activity maximizes specific activity • Improved labeling kinetics maximizes yields • Flexible—both -dATP and -dCTP buffers supplied with each kit • Decamers produce greater primer-template stability
Don't Trade Specific Activity for Yield There is a trade-off between probe yield and probe specific activity when using the random-priming method for labeling DNA. Both limiting nucleotide and template mass affect probe yield. Until the labeled nucleotide becomes limiting, the larger the amount of DNA template used, the greater the yield of probe. However, once the labeled nucleotide becomes limiting, additional template will only result in lower specific activity, since the unlabeled template competes with the labeled probe for target. The accompanying figure shows the effect of probe-specific activity on the limits of target detection. Note that probe made with the smallest amount of template DNA (6.25 ng) was able to detect target present at 1/4 to 1/8 the level as the probe made with the largest amount of template (100 ng).
Maximal Yields of High Specific Activity Probes The DECAprime™ II DNA Labeling Kit produces probes with maximum specific activity even when the DNA template is impure or the quantity is unknown or very low. Data shows that low template amounts require long incubation times to reach maximum specific activity. Under these conditions, extended reaction times will increase both the yield and the specific activity of the probe.
Accessory Products: The NucAway™ Spin Columns (SKU# AM10070) are recommended for probe purification.
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