phi29-XT RCA Kit

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E1603L		500 reactions	$2,030.00		$1,827.00
E1603S		100 reactions	$479.00		$431.10

Catalog #	Size	List Price	Your Price
E1603L	500 reactions	$2,030.00	$1,827.00
E1603S	100 reactions	$479.00	$431.10

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 yield in 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.

E1603 Workflow — 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.

E1603 More Product — 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.

E1603 Efficient Amplification — 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.

E1603 Sensitivity 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.

E1603 RCA Cellfree Expression — 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.

Reagents Supplied

The following reagents are supplied with this product:

NEB #

Component Name

Component #

Stored at (°C)

Amount

Concentration

E1603S -20

phi29-XT DNA Polymerase	M0572SVIAL	-20	1 x 0.2 ml	10 X
phi29-XT Reaction Buffer	B0572SVIAL	-20	1 x 1 ml	5 X
Exonuclease-Resistant Random Primers	S1884SVIAL	-20	1 x 0.2 ml	500 µM
Deoxynucleotide (dNTP) Solution Mix	N0447SVIAL	-20	1 x 0.2 ml	10 mM each dNTP

E1603L -20

phi29-XT DNA Polymerase	M0572LVIAL	-20	1 x 1 ml	10 X
phi29-XT Reaction Buffer	B0572SVIAL	-20	2 x 1 ml	5 X
Exonuclease-Resistant Random Primers	S1884LVIAL	-20	1 x 1 ml	500 µM
Deoxynucleotide (dNTP) Solution Mix	N0447XVIAL	-20	1 x 1 ml	10 mM each dNTP

Properties & Usage

FAQs

Quality Control Assay

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

E1603S_L_v1

Certificate of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety Data Sheets

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