DIG-High Prime

Description

• General description: DIG-High Prime has enzyme and nucleotide mixture for rapid random-primed labeling of DNA with Digoxigenin-11-dUTP. In this method, the complementary DNA strand of denatured DNA is synthesized by Klenow polymerase using the 3′-OH termini of the random oligonucleotides as primers. DIG-labeled probes are generated at high yield within one hour or after overnight incubation.
• Specificity: Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or by heating to 65 °C for 10 minutes.
• Application: DIG-High Prime-labeled DNA probes has been used in a variety of hybridization techniques : in Southern blots[1]in northern blotsin dot/slot blotsfor screening of gene librariesin In situ hybridizations[2]Due to highly specific and sensitive detection systems, DIG-labeled DNA probes can be used for single-copy gene detection in 1μg total human DNA. The use of the alkali-labile form of DIG-dUTP, in which the digoxigenin moiety is connected to the spacer arm via an alkali-labile ester bond, enables easier and more efficient stripping and reprobing of blots.
• Features and Benefits: DIG-High Prime guarantees efficient labeling of:DNA amounts ranging from 10ng to 3μg in a standard reaction.DNA of different lengths ranging from small restriction fragments to λ or cosmid DNA.DNA, supercoiled or linearized.DNA in low melting-point agarose. Labeling efficiency: A standard labeling reaction with 1μg template yields 0.8μg newly synthesized digoxigenin-labeled DNA after 1 hour, and 2μg after a 20-hour incubation at +37°C.Contents5x concentrated random primer mix: 1U/ μl Klenow polymerase, labeling grade, 1mM dATP, 1mM dCTP, 1mM dGTP, 0.65mM dTTP, 0.35mM DIG-11-dUTP, alkali labile in 50% (v/v) glycerol.
• Quality: Function test:In a standard assay with 1μg linearized pBR 328, 0.8μg of DIG-labeled DNA is synthesized after 1 hour, and 2.3μg after 20 hours. When this labeled DNA is used for hybridization at a concentration of 20ng/ml, 0.03pg homologous DNA are detected by chemiluminescence with the anti-DIG-alkaline phosphatase conjugate and CSPD on a dot or Southern blot.
• Principle: DIG-labeled DNA probes are generated with DIG-High Prime according to the random-primed labeling technique. DIG-High Prime is a specifically developed reaction mixture containing Digoxigenin-11-dUTP and all reagents necessary for random-primed labeling, including Klenow enzyme, premixed in an optimized 5x concentrated reaction buffer in 50% glycerol.
• Preparation Note: DIG-labeled probes in the reaction mix or in the hybridization buffer are stable for more than 12 months stored at -15 to -25°C. They can be reused several times if freshly denatured before use.Assay Time: 80 minutesSample Materials DNA fragments of at least 100bpLinearized plasmid, cosmid or λDNASupercoiled DNAOr minimal amounts of DNA (10ng), e.g., DNA restriction fragments isolated from gels or in molten agaroseNote: To obtain optimal results, template DNA should be linearized and should have a size of = 100bp or larger. Template DNA > 5kb should be restriction-digested using a 4bp cutter prior to labeling.
• Other Notes: For life science research only. Not for use in diagnostic procedures.