Application

For convenient and efficient generation of DIG-labeled DNA probes in polymerase chain reaction (PCR). DIG-labeled hybridization probes are highly sensetive and suitable for low-(single)-copy gene detection of rare mRNA in Southern and northern blots.
The kit contains the Expand High Fidelity DNA polymerase mix. This robust enzyme mix with proofreading activity will polymerize probes 40 bp to 5 kb long using 10 pg plasmid DNA and 10 ng genomic DNA as template.
The concentration of the supplied dUTP-nucleotide mix can be adjusted according to probe length. Labeling effectiveness can quickly be determined on an agarose gel.
Stripping and reprobing of membranes is possible multiple times following the protocol in the package insert.
One PCR labeling reaction (50 μl) will typically yield enough probe for 20 ml hybridization solution. The kit can be used for approximately 25 reactions (50 μl). DIG-labeled probes are stable for over one year.

General description

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Kit for the generation of highly sensitive hybridization probes, that are suitable for the detection of low- (single-) copy target sequences. Probes are labeled with DIG-dUTP (alkali-labile), by the method of polymerase chain reaction. The ratio of DIG-dUTP:dTTP is 1:2.

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

Sample Materials?
Any DNA suitable as PCR template can be labeled. Use either:
??Plasmid DNA, 10 to 100pg (optimal amount, 10pg)
??Genomic DNA, 1 to 50ng (optimal amount, 10ng)

Template concentration during PCR is the most critical factor in producing specific probes. For most templates, use no more than the amounts given above. Too much template will lead to coamplification of primary extension products (those copied past the priming sites). These primary extension products may contain repetitive sequences or unrelated products from secondary priming sites (if prepared from genomic DNA) or vector sequences (if prepared from plasmid DNA). In subsequent hybridization assays, the probe-target hybrid will be a smear because the probe will cross-hybridize with vector or genomic DNA sequences.
For best results, use cloned inserts as template. Genomic DNA can be more difficult to use.
Purity of template is not as critical for PCR labeling as for other types of labeling.

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

One reaction can produce enough labeled probe to analyze 650cm²?of blot membrane.

Packaging

1 kit containing 6 components.

Quality

Each lot of the PCR DIG Probe Synthesis Kit is function tested in PCR. Amplification products are assayed in genomic Southern blots.
Under PCR conditions as described in this package insert, the control reaction generates an amplification product of 442bp. Due to multiple incorporations of DIG-dUTP during the PCR process, the molecular weight of the PCR products is significantly increased compared to the unlabeled PCR product. A specific fragment pattern is detected after hybridization of the PCR product to 10μg human genomic DNA followed by chemiluminescent detection.