Storage: −20°C
UNSPSC Code: 41106000
Components: 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
RIDADR: NONH for all modes of transport
Application:
For convenient and efficient generation of DIG-labeled DNA probes in polymerase chain reaction (PCR). DIG-labeled hybridization probes are highly sensitive and 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 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:
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:
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.