Product Class: Kit

phi29-XT RCA Kit


Catalog #E1603

Product Introduction

  • Powered by phi29-XT DNA Polymerase, an engineered polymerase that generates high product yield in a short reaction time with improved thermostability and sensitivity
  • Amplify from as little as 1 fg of circular DNA input
  • Flexible protocol offers compatibility with different types of sample input material
  • Enables multiple downstream applications without further processing steps
  • Kit includes dNTPs and exonuclease-resistant random primers
  • View the Enhanced Rolling Circle Amplification Performance with a Newly Engineered phi29 DNA Polymerase NEB TV webinar

Product Information

Description

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. 

Figure 1: Overview of the phi29-XT RCA Kit

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.



Figure 2: phi29-XT DNA Polymerase generates more product in less time than wild-type phi29 DNA Polymerase, at elevated temperatures

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.



Figure 3: The phi29-XT RCA Kit efficiently amplifies as little as 1 fg pUC19 DNA

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.



Figure 4: The phi29-XT RCA Kit offers exceptional sensitivity and product yield

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.



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

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.



This product is related to the following categories:
Isothermal Amplification & Strand Displacement Products
This product can be used in the following applications:
Isothermal Amplification

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Protocols for amplification of DNA using the phi29-XT RCA Kit (NEB #E1603)
  2. General Protocols for Downstream Applications (NEB #E1603)

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the advantage of using phi29-XT DNA Polymerase instead of wild-type phi29?
  2. How can I increase the RCA yield?
  3. At what temperature range can the phi29-XT RCA Kit be used?
  4. Can the phi29-XT RCA Kit be used to generate single-stranded DNA?
  5. Can the phi29-XT RCA Kit be used to amplify large, circular DNA constructs such as BACs and Fosmids?
  6. Does the input DNA template have to be circular?
  7. How can I verify successful amplification and specificity of my RCA reaction?
  8. How can I debranch my RCA products?
  9. Do I need to purify the RCA products prior to downstream applications?
  10. How do I quantify my RCA products?
  11. Can phi29-XT DNA Polymerase be heat inactivated?
  12. Can I use this kit to incorporate 5-methyl-dCTP?
  13. Does phi29-XT DNA Polymerase work in wild-type phi29 DNA Polymerase Reaction Buffer?
  14. Can phi29-XT DNA Polymerase be used in wild-type phi29 RCA reaction conditions (1X phi29 DNA Polymerase Reaction Buffer at 30°C)?
  15. Use of specific primers for RCA with phi29-XT
  16. What is the difference between the phi29-XT WGA Kit (NEB #E1604) and the phi29-XT RCA Kit (NEB #E1603)?

Quality, Safety & Legal

Quality Assurance Statement

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]

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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