Not available outside of the UK & Ireland.

Application

KAPA2G Fast Genotyping Mix:has been used for the extraction and amplification from mouse tail, ear, and toe tissuehas been used for the extraction and amplification of DNA from other animal tissue and hyridomasis suitable for use in polymerase chain reaction (PCR)

Biochem/physiol Actions

DNA extracted with KAPA Express Extract is amplified with KAPA2G Fast Genotyping Mix with dye. DNA fragments generated with KAPA2G Fast Genotyping Mix are 3’;-dA-tailed and may be cloned into TA cloning vectors, or used for routine downstream analyses or applications, including restriction enzyme digestion, cloning and sequencing. Like wild-type Taq, KAPA2G Fast has 5’;→3’; polymerase and 5’;→3’; exonuclease activities, but no 3’;→5’; exonuclease (proofreading) activity. The fidelity of KAPA2G Fast is like that of wild-type Taq; it has an error rate of approximately 1 error per 1.7 x 10⁵ nucleotides incorporated. Lysis is performed in a thermocycler, heating block or water bath, after which the sample is centrifuged to recover the DNA containing supernatant. The supernatant is then diluted 10-fold before use as a template in PCR. Unlike existing protocols that rely on Proteinase K digestion, extractions using KAPA Express Extract are conveniently performed in a single tube, without the need for hazardous chemicals, or multiple wash steps. This greatly reduces the risk of sample loss and contamination. The process yields enough template for multiple assays and is easily scaled to handle samples in 96-well format. Each extraction yields enough template for at least 100 genotyping PCR reactions. Extracts (either diluted or undiluted) are stable at -15°C to -25°C for at least 6 months.

Features and Benefits

Increased throughput, turnaround time and reliability: PCR-ready DNA generated in as little as 15 minutes Minimal handling, thereby reducing the risk of sample loss or contamination Sufficient template for multiple assays; easily scaled to handle samples in a 96-well format Fast and reliable amplification of DNA fragments across a wide range of amplicon lengths and GC contentsOutperforms crude extraction methods and commercial kits: Increased turnaround times Increased specificity Decreased costQuick Notes: Extract PCR-ready DNA from mouse tail, ear, or toe tissue in 15 minutes.Dilute DNA extract 10-fold prior to PCR in 10 mM Tris-HCl (pH 8.0–8.5), and use 1 µL of DNA per 25 µL reaction. Extracts are stable at -20°C for >6 months. KAPA2G Fast (HotStart) Genotyping Mix contains the engineered KAPA2G Fast DNA Polymerase for fast PCR, along with buffer, MgCl₂ (1.5 mM at 1X), and dNTPs (0.2 mM each at 1X). Loading dye for electrophoresis is included in the KAPA2G Fast Genotyping Mix, which allows direct loading of PCR products onto agarose gels for analysis. Use 15 sec annealing time, with 15 sec extension time for amplicons ﹤1 kb, or 15–30 sec/kb extension for longer amplicons.

General description

KAPA2G Fast Genotyping Mix (2X) is a ready-to-use master mix containing all components for fast polymerase chain reaction (PCR), except primers and template. The ReadyMix^™ also contains two inert dyes, allowing for the analysis of PCR reaction products by agarose gel electrophoresis without the need to add a DNA loading solution. The KAPA Express Extract system contains a thermostable protease, designed for rapid sample lysis. The KAPA Express Extract, a novel thermostable protease and buffer system allows the extraction of PCR-ready DNA from mouse tissue in as little as 15 minutes, and KAPA2G Fast Genotyping Mix with dye, containing a DNA polymerase engineered via directed evolution for high processivity and extreme speed. The combination of KAPA Express Extract and KAPA2G Fast Genotyping Mix allows for the reliable extraction and amplification of DNA fragments from mouse tissue in as little as 1 hour, as compared to ≥1 day with conventional protocols.

Legal Information

ReadyMix is a trademark of Sigma-Aldrich Co. LLC

Other Notes

For Research Use Only. Not for use in diagnostic procedures.

Preparation Note

Always ensure that the product has been fully thawed and mixed before use. Reagents may be stored at 4°C for short-term use (up to 1 month). Return to -20°C for long-term storage. Provided that all components have been handled carefully and not contaminated, the kit is not expected to be compromised if left (unintentionally) at room temperature for a short period of time (up to 3 days).

Quality

All components contained with KAPA Mouse Genotyping Kits are subjected to stringent quality control tests, are free of contaminating exo- and endonuclease activities and meet strict requirements with respect to DNA contamination.