KAPA2G Fast Genotyping Mix

The Uses of KAPA2G Fast Genotyping Mix

KAPA2G Fast Genotyping Mix:

• has been used for the extraction and amplification from mouse tail, ear, and toe tissue
• has been used for the extraction and amplification of DNA from other animal tissue and hyridomas
• is suitable for use in polymerase chain reaction (PCR)

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.

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.