Application

KAPA2G Robust HotStart^® ReadyMix^™ has been used for the:amplification of GC- or AT-rich templatesaplification of templates containing common polymerase chain reaction (PCR) inhibitors e.g. salts, urea, SDS, or ethanolcrude sample PCR e.g. buccal swabs, cultured mammalian, yeast, or bacterial cellscolony PCRDNA extraction and amplificationmutation screeningmethylation-specific PCR (MSP) amplificationgenotyping-PCRPCR-based sequencing

Biochem/physiol Actions

DNA fragments generated with KAPA2G Robust HotStart^® DNA Polymerase have the same characteristics as DNA fragments generated with wild-type Taq DNA polymerase and may be used for routine downstream analyses or applications, including restriction enzyme digestion, cloning, and sequencing. Like wild-type Taq, KAPA2G Robust HotStart has 5’;→3’; polymerase and 5’;→3’; exonuclease activities, but no 3’;→5’; exonuclease (proofreading) activity. The fidelity of KAPA2G Robust HotStart is similar to that of wild-type Taq; it has an error rate of approximately 1 error per 1.7 x 105 nucleotides incorporated. PCR products generated by KAPA2G Robust HotStart are 3’;-dA-tailed and may be cloned into TA cloning vectors.

Features and Benefits

Efficient amplification of GC- and AT-rich targets Robust performance across a wide range of GC- and AT-rich templates Increased PCR success ratesImproved tolerance to common PCR inhibitors Efficient amplification from crude samples Higher yield and sensitivity per unit of enzymeUnrivalled performance in colony PCR Higher yields and improved consistency of PCR direct from E. coli and yeast cellsQuick Notes: KAPA2G Robust HotStart ReadyMix PCR Kits contain KAPA2G Robust DNA Polymerase, engineered for high processivity and inhibitor tolerance. Both purified genomic DNA and crude samples (e.g. colony PCR) can be used as template. Use 15 sec/kb extension time per cycle, and increase to 30-60 sec/kb for difficult amplicons or templates. KAPA2G Robust HotStart ReadyMix contains 2 mM MgCl₂ at 1X. KAPA2G Robust HotStart ReadyMix with dye includes two inert tracking dyes to allow direct loading onto agarose gels. KAPA2G Robust HotStart ReadyMix is ideal for single-protocol PCR, i.e. amplification of fragments varying from 25–65% GC, up to 1 kb in size, using a single reaction setup and cycling protocol. Reaction products are 3’;-dA-tailed and may be cloned into TA cloning vectors.

General description

The second-generation KAPA2G Robust DNA Polymerase was evolved to solve inconsistent amplification across a broad range of amplicon types (GC- and AT-rich). KAPA2G Robust HotStart^® ReadyMix^™ enables higher processivity and specific activity, which translates to robust PCR performance, high sensitivity, and improved tolerance to common inhibitors. The high performance of the KAPA2G Robust DNA Polymerase is ideally suited for challenging PCR applications and difficult samples, eliminating the need for optimization using multiple enzymes and protocols.

Legal Information

ReadyMix is a trademark of Sigma-Aldrich Co. LLC

HOTSTART is a registered trademark of Molecular BioProducts, Inc.

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). Long-term storage at room temperature and 4°C is not recommended. Please note that reagents stored at temperatures above -20°C are more prone to degradation when contaminated during use, and therefore storage at such temperatures is at the user′s own risk.

Quality

Each batch of KAPA2G Robust HotStart DNA Polymerase is confirmed to contain ﹤2% contaminating protein (Agilent Protein 230 Assay). KAPA2G Robust HotStart ReadyMix PCR Kits are subjected to stringent quality control tests, are free of contaminating exo- and endonuclease activity, and meet strict requirements with respect to DNA contamination levels.