The Ambion® KinaseMax™ 5' End-Labeling Kit allows the efficient end-labeling of DNA or RNA to high-specific activity with T4 polynucleotide kinase and [gamma-32P] ATP, or quantitative phosphorylation of 5' ends using unlabeled ATP. Includes sufficient reagents for 30 reactions.
• Label oligonucleotide probes for nuclease protection assays and blot hybridizations
• Label primers for northerns and quantitative PCR
• Obtain up to 3-fold greater yields than with standard kinase buffers
• Reagents included for forward and dephosphorylation reactions
The kit includes a kinase reaction buffer that exceeds the performance of the standard forward reaction buffers recommended in "Molecular Cloning: A Laboratory Manual" (Sambrook et al., 1989) and "Current Protocols in Molecular Biology" (John Wiley & Sons, Inc. Ausubel, FM et al. editors, 1994). Crude 7000 Ci/mmol [gamma-32P]ATP is compatible with this kit. End-labeled probes are more stable than internally labeled probes and can routinely be used in assays for two to four weeks.
Rapid, Phenol-Free Phosphatase Removal Step
Inactivating the Calf Intestinal Phosphatase (CIP) after the dephosphorylation reaction is a time-consuming and cumbersome procedure that usually requires phenol extraction and ethanol precipitation typically resulting in sample loss. The KinaseMax™ Kit now includes a novel Phosphatase Removal Reagent for quick and complete removal of CIP. After the dephosphorylation step, the reagent is added to the reaction, incubated at room temperature for 2 minutes. The tube is then spun for a minute in the microfuge and the supernatant transferred to a new tube for the kinasing reaction.
Accessory Products:
The NucAway™ Spin Columns (SKU# AM10070) are available for rapid purification of end-labeled probes.
Contents and Storage
T4 Polynucleotide Kinase, Calf Intestine Alkaline Phosphatase, 10X Kinase Buffer, Dephosphorylation Buffer, Phosphatase Removal Reagent, RNA Transcript Control, pUC19⁄Sau3 A Markers, ATP Solution, Gel Loading Buffer II should be stored at –20°C. Nuclease-free water may be stored at any temperature.
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