Product Class: Kit

phi29-XT WGA Kit


Catalog #E1604

Product Introduction

  • Powered by phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability and sensitivity
  • High sensitivity: amplify from as little as 10 fg of input DNA
  • Flexible protocol offers compatibility with different types of sample input material
  • Simple workflow produces high yield in a short reaction time (less than 2 hours)
  • Whole genome representation for human and microbial DNA
  • Enables multiple downstream applications without further processing steps
  • View the Enabling Efficient Whole Genome Amplification with a Newly Engineered phi29 DNA Polymerase NEB TV webinar

E1604 Time Temp

Product Information

Description

The phi29-XT WGA Kit is optimized for sensitive, fast, and robust whole genome amplification (WGA), an isothermal method for amplification of an entire genome, starting from femtogram quantities of DNA and resulting in microgram quantities of amplified products. The kit features phi29-XT DNA Polymerase, an engineered polymerase with improved thermostability, sensitivity, and capable of generating a higher yield in a shorter reaction time than wild-type phi29 DNA Polymerase. Also included are exonuclease-resistant random primers (containing phosphorothioate bonds) to universally amplify any DNA sequence, neutralization buffer, and dNTPs. Input material can be purified genomic DNA, cells, or microbiomes. WGA enables several downstream applications, including single cell whole genome sequencing (WGS), genomic DNA enrichment, and environmental DNA (eDNA) enrichment and sequencing.

Figure 1: Overview of the phi29-XT WGA Kit

E1604 Workflow


The phi29-XT WGA Kit (NEB #E1604) is a fast, easy-to-use, and highly versatile kit containing all the required components for whole genome amplification (WGA) using a random primer mix. The kit delivers high yields of DNA products from a variety of starting materials including purified genomic DNA, cells, or microbiomes. This kit is ideal for various DNA applications such as single cell whole genome sequencing (WGS), genomic DNA enrichment, and environmental DNA (eDNA) enrichment and sequencing.


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

E1604 More Product Less Time

Triplicate WGA reactions were performed with 100 pg human genomic DNA input at the indicated temperatures for 1.5 hours. The reactions were then heat-inactivated 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 for WGA and 1X phi29-XT Reaction Buffer for WGA. Reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (lines) to determine the yield at each reaction temperature. While the working reaction temperature of wild-type phi29 DNA Polymerase is typically between 30°C and 38°C, phi29-XT DNA Polymerase generates robust product yields around 42°C. The optimal temperature range for wild-type phi29 increased by approximately three degrees for WGA using human genomic DNA when compared to RCA using pUC19 plasmid as input.


Figure 3: phi29-XT DNA Polymerase generates high yields of product in a short period of time, even from 10 fg genomic DNA input

E1604 High Yield

Duplicate WGA reactions were carried out using various amounts of human genomic DNA as input and were incubated for 0.5 to 8 hours at 42°C, followed by heat inactivation at 65°C for 10 minutes. All reactions contained 1 mM dNTPs and 50 μM Exonuclease-Resistant Random Primers. The resulting reaction yields (dots) were quantified using Quant-iT® PicoGreen® dsDNA Reagent and were averaged (line) to determine the yield at each reaction temperature. While nonspecific amplification will eventually occur in the absence of template from primer-dimer amplification, there is no NTC amplification within the recommended incubation period of 1.5 h. Nonspecific, primer-dimer amplification is not expected to significantly contribute to the WGA yield when DNA input is included in the reaction. NTC = no template control.


Figure 4: phi29-XT WGA Kit can be used with a variety of input sources, consistently yielding more than 10 μg of DNA

E1604 Input Sources

Bar chart showing the average yield (μg) of triplicate reactions from various input sources. Samples were incubated at 42°C for 1.5 hours, then heat-inactivated at 65°C for 10 minutes. All WGA reactions contained 1 mM dNTPs and 50 μM Exonuclease-Resistant Random Primers. DNA yield is quantified using Quant-iT® PicoGreen® dsDNA Reagent (Invitrogen®, #P11496). Input amounts are listed below each sample type. With the exception of the human cell sample, DNA used as input was extracted from the source prior to WGA.


Figure 5: phi29-XT WGA Kit ensures whole genome coverage for human genomic DNA

E1604 Coverage

Integrative Genome Viewer (IGV) visualization of read coverage across the human genome (human NA19240 gDNA). Libraries were constructed using the NEBNext® Ultra II FS DNA Library Prep Kit for Illumina (NEB #E7805) and sequenced on a NovaSeq® instrument (2 x 150 bp). Coverage depth per base was determined, reads were down-sampled with seqtk and aligned to the human reference genome (NCBI, GRCh38) with Bowtie2. As previously reported, drops in coverage occurred in centromeric regions, which are known for their highly repetitive nature and pose an amplification challenge for DNA polymerases.


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

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E1604S     -20    
      phi29-XT DNA Polymerase for WGA M0574SVIAL -20 1 x 0.05 ml 10 X
      phi29-XT Reaction Buffer for WGA B0574SVIAL -20 1 x 0.5 ml 5 X
      Neutralization Buffer B0575SVIAL -20 1 x 0.5 ml Not Applicable
      Exonuclease-Resistant Random Primers S1884AVIAL -20 1 x 0.05 ml 500 µM
      Deoxynucleotide (dNTP) Solution Mix N0447AVIAL -20 1 x 0.1 ml 10 mM each dNTP
  • E1604L     -20    
      phi29-XT DNA Polymerase for WGA M0574LVIAL -20 1 x 0.2 ml 10 X
      phi29-XT Reaction Buffer for WGA B0574SVIAL -20 1 x 0.5 ml 5 X
      Neutralization Buffer B0575SVIAL -20 1 x 0.5 ml Not Applicable
      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

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Protocol for amplification of DNA using the phi29-XT WGA Kit (NEB #E1604)
  2. General Protocols for Downstream Applications (NEB #E1604)

Manuals

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

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. How can I increase the WGA yield when using the phi29-XT WGA Kit (NEB #E1604)?
  2. At what temperature range can the phi29-XT WGA Kit (NEB #E1604) be used?
  3. What is the difference between the phi29-XT WGA Kit (NEB #E1604) and the phi29-XT RCA Kit (NEB #E1603)?
  4. Is the phi29-XT WGA Kit (NEB #E1604) suitable for amplification of short DNA fragments?
  5. When using the phi29-XT WGA Kit (NEB #E1604) do I need to purify the WGA products prior to downstream applications?
  6. How do I quantify my WGA products generated using the phi29-XT WGA Kit (NEB #E1604)?
  7. Can phi29-XT DNA Polymerase be heat inactivated?
  8. When using the phi29-XT WGA Kit (NEB #E1604) can the reaction volume be scaled up or scaled down?
  9. Is the phi29-XT WGA Kit (NEB #E1604) suitable for single cell DNA amplification?
  10. Do I need to extract DNA from whole cells prior to using the phi29-XT WGA Kit (NEB #E1604)?

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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