Application

For Random-primed labeling of DNA probes with DIG-11-dUTP, alkali-labile. DIG-labeled DNA probes can be used for:?
??All types of filter hybridization according to our standard protocol given in the pack insert of the special hybridization solution DIG Easy Hyb.
??Single-copy gene detection in total genomic DNA, even from organisms with high complexity, for example,?human, barley, and wheat.
??In situ?hybridizations.

General description

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Convenient kit for the labeling of DNA with Digoxigenin-dUTP using random oligonucleotides as primers. 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.

Packaging

1 kit containing 7 components.

Principle

DIG-labeled DNA probes are generated according to the random-primed DNA labeling method which is based on the hybridization of random oligonucleotides to the denatured DNA template. The complementary DNA strand is synthesized by Klenow enzyme which uses the 3′-OH termini of the random oligonucleotides as primers and a mixture of deoxyribonucleotides containing DIG-11-dUTP, alkali-labile for elongation. This results in incorporation of digoxigenin into the newly synthesized DNA.

Note:
??The use of the alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization experiments with a second DIG-labeled probe.
??DNA probe, labeled with DIG-11-dUTP, alkali-labile must not be denatured using NaOH, but can be denatured by boiling in a waterbath.?

Quality

Function tested in a 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.

Specifications

Assay Time: Labeling: 1hour to O/N
Sensitivity and specificity:?A single-copy human gene (tPA gene) is detected with a DIG-labeled probe in a Southern blot of 1μg digested human placenta DNA.