Storage: 2-8°C

UNSPSC Code: 41116133

Components: Denaturation Solution for standard protocol; Denaturation Solution for alternate protocol; Hybridization Buffer; Washing Buffer Tablets; Conjugate Dilution Buffer; Anti-Digoxigenin-Peroxidase Conjugate antibody, lyophilizate; ABTS Solution; StreptaWell, microplate modules (8 wells each) in a strip frame; Adhesive Plate Cover Foils

RIDADR: NONH for all modes of transport

Features and Benefits:
This kit is part of a complete PCR ELISA system. If you combine it with other reagents and equipment, you can adapt the system for many molecular biology applications. For example:
€¢ If you have an appropriate target-specific, biotin-labeled capture probe, you can distinguish PCR products that differ by as little as a single base pair. Thus the PCR ELISA system (DIG-labeling + DIG-detection) can be used:
€¢ To detect point mutations, deletions or insertions.
€¢ To classify target sequences (e.g., as in HLA-typing or cell typing).
€¢ Preparation of suitable DIG-labeled standards and addition of a colorimetric detection system will allow you to quantify PCR products.
€¢ The microplate format used by the PCR ELISA system makes the system compatible with automated plate preparation and reading systems.
Labeling: The DIG-labeled PCR products detected with this kit contain alkali-stable digoxigenin-11-dUTP (DIG-dUTP). Such DIG-labeled products can be prepared with the PCR ELISA, DIG-Labeling kit.
Capacity: This size of the PCR ELISA, DIG-Detection kit allows semi-quantitative detection of more DIG-labeled PCR products. The total number of PCR products detected will vary depending upon the number of sample dilutions, controls, and/or standards used in the assay.

Application:
This kit is used in research studies for the semi-quantitative detection of digoxigenin-labeled PCR products. It is particularly suitable for the parallel testing of a large number of samples.
Note: The PCR ELISA system provides a convenient, nonradioactive method for labeling and detecting PCR products. It is approximately 100 times more sensitive than conventional ethidium bromide staining of products on agarose gels.

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.

Specificity:
The high specificity of digoxigenin detection permits distinguishing specific and nonspecific amplification products. This is due to the hybridization step with the capture probe. The specificity of this hybridization follows the standard rules of hybridization assays. Stringent hybridization in the digoxigenin detection depends on the length and the GC content of the capture probe and the incubation temperature. Temperatures up to 55 °C are compatible with digoxigenin detection. Under these conditions, with short capture probes (18 to 20mers), identification of a single base mismatch in the capture oligonucleotide is possible. For this reason, mutations in template DNAs can also be detected using DIG.

Principle:
Step 1: Amplification of DNA in the presence of digoxigenin-11-dUTP (DIG-dUTP) to generate DIG-labeled PCR product.
Step 2: Denaturation of PCR product and hybridization to a biotin-labeled capture probe. The capture probe specifically recognizes an internal sequence in the target DNA.
Step 3: Immobilization of biotin-labeled hybrid on a streptavidin-coated microplate, followed by plate washes to remove unhybridized, non-specific amplification products.
Step 4: Detection of bound hybrids with peroxidase-conjugated anti-digoxigenin antibody (Anti-DIG-POD) and the colorimetric peroxidase substrate, ABTS.
Results can be measured with a standard microplate reader.