Storage: −20°C
UNSPSC Code: 41106000
RIDADR: NONH for all modes of transport
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.
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 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.
General description:
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.
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.
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.
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.
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.