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Phi29 WGA Kit

Phi29 DNA Polymerase-based Whole Genome Amplification

Cat. No. Amount Price (EUR) Buy / Note
APP-004S 25 reactions x 20 μl 126,88 Add to Basket/Quote Add to Notepad
APP-004L 100 reactions x 20 μl 380,63 Add to Basket/Quote Add to Notepad

For research use only!

Shipping: shipped on dry ice

Storage Conditions: store at -20 °C (except control template - store at 4 °C)
avoid freeze/thaw cycles

Shelf Life: 12 months

Description:
Phi29 WGA Kit contains all reagents for representative whole genome amplification (WGA) from limited amounts of purified genomic DNA (gDNA) with Phi29 DNA polymerase. Up to 5-10 μg of amplified gDNA can be obtained from 10-50 ng of purified gDNA starting material within 90 minutes. The average product length is >10 kb with a major band at 10-12 kb and a range between 2 kb and approximately 100 kb. The resulting amplified gDNA can directly be used for down-stream applications such as DNA probe labeling (gDNA fragmentation followed by Klenow fragment-based labeling), PCR or qPCR.

Content:

ComponentAPP-004SAPP-004L
8x Denaturation Buffer25 μl100 μl
10x Neutralization Buffer50 μl200 μl
1x Reaction Buffer300 μl1.2 ml
Phi29 Enzyme Mix25 μl100 μl
1 mM Exo-resistant Random Hexamer Primer
NpNpNpNpSNpSN
25 μl100 μl
10 mM dNTP Mix
Premix of 10 mM dATP, dCTP, dTTP and dGTP
50 μl200 μl
Control template
human mixed genomic DNA
2.5 μg7 μg
PCR-grade H2O 1.2 ml2x 1.2 ml



Amplification principle:
Sequence-independent amplification of gDNA is achieved via an isothermal amplification reaction with Phi29 DNA polymerase and exonuclease-resistant random primer. The unique properties of Phi29 DNA polymerase ensure efficient, minimal bias amplification:

  • strand displacement activity allows simultaneous DNA replication from multiple sites (multiple displacement amplification (MDA))
  • high processivity allows efficient synthesis of DNA fragments with an average length of 10 – 12 kp (maximum up to 100 kb).
  • 3’-5’ exonuclease proofreading activity ensures a 100-fold higher fidelity compared to Taq polymerase.

IMPORTANT:

  • Store all reagents on ice until use and prechill reaction tubes.
  • Work sterile and use filter tips to avoid DNA contamination of stock solutions and samples.

1. Preparation of working solutions

1.1 1x Denaturation and 1x Neutralization Buffer solution

  • Dilute 8x Denaturation and 10x Neutralization Buffer with PCR-grade water, vortex and spin-down briefly
  • One WGA reaction (see 2.1) requires 1 μl of 1x Denaturation and 2 μl of 1x Neutralization buffer.
  • Store dilutions on ice until further use (see 2.1).
  • Prepare dilutions freshly for every experiment. Do not store for subsequent use.

Preparation of 1x Denaturation Buffer for 20 reactions:

CompoundConcentration of stock solutionAmountFinal concentration
Denaturation Buffer8x2.5 μl1x
PCR grade H2O17.5 μl
Total Volume20 μl


Preparation of 1x Neutralization Buffer for 20 reactions:

CompoundConcentration of stock solutionAmountFinal concentration
Neutralization Buffer10x4 μl1x
PCR grade H2O36 μl
Total Volume40 μl

1.2 Preparation of genomic control DNA solution

  • Adjust genomic DNA solution to 100 ng/μl with PCR-grade H2O and store at 4°C.
  • Dilute genomic control DNA solution with PCR-grade H2O to as desired (e.g. 10 ng/μl) and pipet up and down 2-3 times.
  • Store on ice until further use (see 2.2).
  • Prepare dilution freshly for every experiment. Do not store for subsequent use.

2. General protocol for amplification of purified genomic DNA
The protocol below is intended as a general guideline however, individual optimization might be required e.g. in terms of template amount, denaturation conditions or incubation time.

For direct amplification e.g. from blood, buffy coat or buccal swab, please refer to reference [1] and [2] as guideline for sample preparation.

2.1 Genomic template denaturation

  • Transfer 1 μl DNA sample (>/= 10 ng/μl in water or TE buffer) to a prechilled DNAse/RNAse free reaction tube (e.g. 1.5 ml Eppendorf tube).
  • Add 1 μl of 1x Denaturation Buffer (see 1.1) and pipet up and down 2-3 times.
  • Incubate for 3 min at room temperature (15-25°C).
  • Add 2 μl for 1x Neutralization Buffer and pipet up and down 2-3 times.


2.2 Isothermal Whole genome Amplification

Please note: Store all components except of Phi29 Enzyme Mix on ice until use (do not allow them to warm up).

  • Store Phi29 Enzyme Mix at -20°C and take out immediately before use. Prechill reaction tubes on ice.
  • Prepare WGA Master Mix on ice immediately before use by adding all compounds exactly in the order described below:


CompoundFinal conc./amount1 reaction10 reactions
1x Reaction Buffer1x12 μl120 μl
10 mM dNTP Mix1 mM2 μl20 μl
1 mM Exo-resistant Random Hexamer Primer50 μM1 μl10 μl
Phi29 Enzyme Mix1x1 μl10 μl
Total volume16 μl160 μl

  • Vortex and spin-down briefly.
  • Add 16 μl WGA Master Mix to 4 μl of denatured genomic DNA (from 2.1, stored on ice). Total reaction volume: 20 μl.
  • Vortex and spin-down briefly.
  • Incubate for 1.5h at 30°C (e.g. Thermomixer, do not use a water-bath).
  • Inactivate the enzyme for 3 min at 65°C.
  • Proceed with downstream application (e.g. PCR (10-20 fold dilution with TE buffer, use 1-3 μl of diluted DNA) or DNA Labeling). Alternatively, store amplified DNA at -20°C until usage and avoid repeated freeze-thaw-cycles.


2.3 Evaluation of amplified DNA

  • Integrity of amplified DNA can be determined via agarose gel electrophoresis (1-2 μl reaction mix plus DNA intercalator such as EtBr, 1% agarose gel (TAE Buffer)). The average product length should be > 10 kbp with a major band around 10-12 kbp.
  • Quantification of amplified DNA can be performed via fluorescence measurement using Quant-iTTM PicoGreen dsDNA Assay Kit which selectively detects double-stranded DNA (ds DNA) in the presence of ssDNA and free nucleotides.

Selected References:
[1] Dean et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. Proc. Natl. Acad. Sci 99 (8):5261.
[2] Hosono et al. (2003) Unbiased whole-genome amplification directly from clinical samples. Genome Research 13:954.