Application

DIG-High Prime is used for the highly efficient random-primed labeling of DNA probes with DIG-11-dUTP, alkali-labile. DIG-labeled probes are generated at high yield within one hour or after overnight incubation. DIG-High Prime-labeled DNA probes are used in a variety of hybridization techniques: ??Southern blots
??Northern blots
??Dot/slot blots
??Screening of gene libraries
??In situ?hybridizations
Due to highly specific and sensitive detection systems, DIG-labeled DNA probes can be used for single-copy gene detection in 1 μg 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.

Contents
5x 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.

General description

Download the CofA, SDS and IFU specific to your product lot and format

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.

Sigma Life Science is committed to bringing you Greener Alternative Products, which adhere to one or more of The 12 Principles of Greener Chemistry. This product is designed as a safer chemical.? The DIG System was established as a sensitive and cost-effective alternative to using radioactivity for the labeling and detection of nucleic acids. There are many available publications that prove the versatility of the DIG System, so use of radio-labeling is no longer the only option for labeling of DNA for hybridization.

Other Notes

For life science research only. Not for use in diagnostic procedures.

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 minutes

Sample Materials?
??DNA fragments of at least 100bp
??Linearized plasmid, cosmid or λDNA
??Supercoiled DNA
??Or minimal amounts of DNA (10ng), e.g., DNA restriction fragments isolated from gels or in molten agarose

Note:?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.

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.

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.