A single- or double-stranded circular DNA amplification kit with less bias and more uniform amplification starting with limiting amounts of DNA or bacterial cells.
The TruePrime™ RCA kit uses a novel multiple displacement amplification method based on the combination of the recently discovered DNA primase TthPrimPol and the highly processive and high-fidelity Phi29 DNA polymerase to amplify single- or double-stranded circular DNA molecules by rolling circle amplification (RCA). The strong strand displacement capacity of Phi29 DNA polymerase allows TthPrimPol to generate new primers on the displaced strands that are extended by Phi29 DNA pol, resulting in exponential isothermal DNA amplification. The product of TruePrime™ RCA reaction is high molecular weight, double-stranded concatemers of the circular template. The TruePrime™ RCA kit eliminates the need for overnight cell culture and conventional DNA purification. Typical DNA yields from a 25 µL reaction are >3 µg for a 3 hour reaction when starting from 1 ng of input plasmid DNA (3-10 kb). For 150-200 kb templates, yields are approximately 2 µg per 25 µL reaction with 10 ng DNA and a 3 hour reaction.
Suitable starting materials include:
Typical DNA yields from a 25 µL reaction are >3 µg from 1 ng of input plasmid DNA (3-10 kb) and a 3 hour reaction. For 150-200 kb templates yields are approximately 2 µg per 25 µl reaction with 10 ng input DNA and a 3 hour reaction. Yields and kinetics will vary if crude or un-quantified samples are amplified. Reactions without input DNA (no template controls) do not produce any amplification product during 3 hour reactions. Amplification products can be transformed after digestion with a single-site restriction enzyme and re-ligation.
Each batch of TruePrime™ RCA kits is tested against predetermined specifications to ensure consistent product quality. Enzymes used in the kit have been tested separately to ensure adherence to specifications.
Shipping and Storage:
Kits are shipped on dry ice. Upon receipt, the kit should be stored immediately at -20°C in a non-frost-free (constant-temperature) freezer. When stored under these conditions and handled correctly, the products can be kept at least six months after shipping without showing any reduction in performance. For longer periods of time, store the kit at -80°C.