11636090910

PCR DIG Probe Synthesis Kit

sufficient for 25 reaction (50 μL final reaction volume)

• Synonym(s): DIG system, probe

Properties

• usage: sufficient for 25 reaction (50 μL final reaction volume)
• Quality Level: 100
• manufacturer/tradename: Roche
• storage temp.: −20°C

Description

General description

The PCR DIG Probe Synthesis Kit contains an alkali-labile digoxigenin (DIG)-11deoxyuridinetriphosphate (dUTP) formulation. This kit is for convenient and efficient generation of DIG-labeled DNA probes that are highly sensitive in polymerase chain reaction (PCR). The probes are labeled with DIG-11dUTP (alkali-labile), by the method of PCR. The ratio of DIG-dUTP:dTTP is 1:2.
The PCR DIG Probe Synthesis 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 DIG-labeled probes are stable for over one year.

Application

Digoxigenin (DIG)-labeled DNA probes produced from the PCR DIG Probe Synthesis Kit is suitable for low-(single)-copy gene detection of rare mRNA in Southern and northern blots. DIG-labeled DNA probes have also been used for labeling of DNA during in situ hybridization.
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).

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.

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.

Kit Components Only

• Enzyme Mix, Expand High Fidelity 3.5 U/μl
• PCR DIG Probe Synthesis Mix, containing dATP, dCTP, dGTP (2 mM each) 10x concentrated
• PCR Buffer with MgCl₂ 10x concentrated
• dNTP Stock Solution, containing dATP, dCTP, dGTP, dTTP (2 mM each), pH 7.0 10x concentrated
• Control Template, plasmid DNA in Tris/EDTA buffer, pH 8.0. The 5-kb plasmid contains the cDNA for the human tissue-type plasminogen activator (tPA) 20 pg/μl
• Control PCR Primer Mix, containing 50 pmol of each primer, control PCR primer 1 and 2 2 mM each

Safety Information

• Hazard Statements: H412
• Precautionary Statements: P273 - P501
• Hazard Classifications: Aquatic Chronic 3
• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 2
• Flash Point(F): does not flash
• Flash Point(C): does not flash