The phi29-XT RCA Kit includes everything needed for sensitive and robust rolling circle amplification (RCA), an isothermal amplification method to continuously amplify circular DNA by generating long, repetitive copies of the circular sequence. The kit features phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability and sensitivity, and generates higher yieldin a shorter reaction time than wild-type phi29 DNA Polymerase. Also included are dNTPs and exonuclease-resistant random primers (containing phosphorothioate bonds) to universally amplify circular DNA sequences. Input material can be purified circular DNA (single or double-stranded) or direct from liquid media culture, agar plate colonies, and glycerol stocks without the need for DNA extraction. RCA products can be used directly in downstream applications such as DNA sequencing, cell-free DNA enrichment, cell-free protein expression, and DNA biosensors.Figure 1: Overview of the phi29-XT RCA KitThe phi29-XT RCA Kit (NEB #E1603) is a fast, simple to use, and highly versatile kit containing all the required components for rolling circle amplification (RCA) using a random primer mix. The kit delivers high yields of DNA products from a variety of starting materials including purified circular DNA or bacterial cells. This kit is ideal for various DNA applications such as DNA sequencing, cell-free DNA enrichment, cell-free protein expression and DNA biosensors.Figure 2: phi29-XT DNA Polymerase generates more product in less time than wild-type phi29 DNA Polymerase, at elevated temperaturesTriplicate RCA reactions were conducted with 0.1 pg pUC19 plasmid input (NEB #N3041) at the indicated temperatures for 2 hours, followed by heat inactivation at 65°C for 10 minutes. All reactions contained 1 mM dNTPs and 50 µM Exonuclease-Resistant Random Primers. Wild-type phi29 reactions were carried out with 10 units of phi29 DNA Polymerase (NEB #M0269) in 1X phi29 DNA Polymerase Reaction Buffer and 0.1 mg/mL Recombinant Albumin, and phi29-XT reactions were carried out with 1X phi29-XT DNA Polymerase and 1X phi29-XT Reaction Buffer. Reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (line) to determine the yield at each reaction temperature. While the working reaction temperature of wild-type phi29 DNA Polymerase is between 30°C and 37°C, phi29-XT DNA Polymerase generates robust product yields around 42°C.Figure 3: The phi29-XT RCA Kit efficiently amplifies as little as 1 fg pUC19 DNAQuadruplicate RCA reactions were carried out with the phi29-XT RCA Kit (NEB #E1603) at 42°C for 2 hours in the absence of template (NTC) or with 1 fg pUC19 plasmid. Reaction products were digested with AclI (NEB #R0598), which site-specifically cuts the plasmid sequence to generate two fragments (373 bp and 2,313 bp), and run on an agarose gel to verify successful amplification. phi29-XT DNA Polymerase supports robust amplification of 1 fg of 2.7 kb circular plasmid DNA in just 2 hours.Figure 4: The phi29-XT RCA Kit offers exceptional sensitivity and product yieldTriplicate RCA reactions were carried out using commercially available phi29 DNA polymerases, according to manufacturers’ protocols, for 2 hours with 1 pg or 1 ng pUC19 plasmid as the starting material. Reaction yields (dots) were quantified using Quant-iT®PicoGreen® dsDNA Reagent and averaged (bar). The phi29-XT RCA Kit (NEB #E1603) generates more product in less time than other commercially available products.Figure 5: Direct colony RCA with the phi29-XT RCA Kit enables Sanger sequencing andcell-free protein expression in less time than a traditional plasmid preparation approachA. A single bacterial colony, harboring a modified pET28a vector with eGFP under a T7 promoter, was picked and diluted in water. Additional plasmid was then produced in vitro using the phi29-XT RCA Kit (NEB #E1603), or by traditional liquid culture using bacterial cells.B. Sanger sequencing was performed on the RCA product (1 µl of 20-fold diluted RCA reaction) or plasmid (900 ng) extracted from the liquid culture, as depicted.C. Following sequence verification, GFP protein was expressed with the NEBExpress® Cell-free E. coli Protein Synthesis System (NEB #E5360) using either 2.4 µl of 20-fold diluted RCA reaction or 100 ng of purified plasmid in 10 µl reaction volume. Protein synthesis was monitored by measuring eGFP fluorescence, highlighting that RCA products enable robust cell-free protein expression of GFP in a shorter amount of time than plasmid prep.
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