Description

TempliPhi Large Construct DNA Amplification Kit (TempliPhi LC Kit) was developed specifically to prepare templates for BAC or fosmid DNA sequencing. The TempliPhi method utilizes bacteriophage Phi29 DNA polymerase enzyme to exponentially amplify single- or double-stranded circular DNA templates by rolling circle amplification (RCA). This isothermal amplification method produces microgram quantities of DNA from picogram amounts of starting material in a few hours. Amplification in vitro of very small amounts of template DNA eliminates the need for overnight cell culture and conventional plasmid or M13 DNA purification. The proofreading activity of Phi29 DNA polymerase ensures high fidelity DNA replication.

The starting material for amplification can be a small amount of bacterial cells containing a BAC or fosmid DNA, or any circular DNA sample. Bacterial colonies can be picked from agar plates and added directly to the TempliPhi LC reaction. Alternatively, microliter quantities of a saturated bacterial culture or a glycerol stock can serve as starting material. Regardless of the source of starting material, amplification is completed in 18 h at 30°C with no needed for thermal cycling. The product of the TempliPhi Large Construct reaction is high molecular weight, double-stranded concatemers of the circular template. The TempliPhi Large Construct product is double-stranded DNA that can be sequenced with forward and reverse primers. DNA amplified by the TempliPhi Large Construct Amplification method can be used directly in cycle sequencing reactions without any purification.