Figure 1: Overview of the phi29-XT RCA Kit

The 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 temperatures

Triplicate 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 DNA

Quadruplicate 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 yield

Triplicate 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 and cell-free protein expression in less time than a traditional plasmid preparation approach

A. 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.