phi29-XT WGA Kit

Catalog #	Concentration	Size	List Price	Quantity	Your Price
E1604L		100 reactions	$957.00		$861.30
E1604S		25 reactions	$307.00		$276.30

Catalog #	Size	List Price	Your Price
E1604L	100 reactions	$957.00	$861.30
E1604S	25 reactions	$307.00	$276.30

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.

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.

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.

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.

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.

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.

Reagents Supplied

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
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
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

An exception occurred during the operation, making the result invalid. Check InnerException for exception details.

Protocols

DataCards

manuale1604

Selection Charts

DNA Polymerase Selection Chart

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]

E1604S_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

Deoxynucleotide (dNTP) Solution Mix

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