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Protocol for GenomePlex Whole Genome Amplification from Soil

Application Guide

I. Description
II. Product Components
III. Materials to be Supplied by the User
IV. Protocol for Extraction of DNA from Soil
V. Protocol for GenomePlex Whole Genome Amplification from Soil
VI. Quantification of Amplified products
VII. Purification of Amplified Products
Appendix
References
Contact Information

Application Guide

I. Description
This protocol addresses the need for extracting, amplifying, and purifying genomic DNA from soil samples. Genomic DNA from the soil samples can be damaged and of low yield. These qualities hurt the researchers ability to perform downstream analysis. GenomePlex WGA provides a method of amplifying nanogram quantities of genomic DNA from soil with little or no detectable bias. The methods described use a soil extraction kit, GenomePlex WGA kit, and GenElute PCR Clean-up Kit.

This technology maintains the genetic representation with concordance >99.8% in genotyping results from comparing genomic DNA and GenomePlex amplified DNA. In addition to SNP genotyping, downstream applications also include performing TaqMan assays and BeadArray analysis.

II. Product Components

  • UltraClean Soil Kit by Mo Bio Laboratories, Inc. (12800-50)
  • GenomePlex Whole Genome Amplification Kit (WGA1)
  • GenElute PCR Clean-Up Kit (NA1020)

III. Materials to be Supplied by the User

  • Soil samples
  • 1.5 ml microcentrifuge tubes for lysis, Product Code T9661
  • Microcentrifuge (with rotor for 2ml tubes)
  • Ethanol (absolute), Product Code E7023
  • Molecular Biology Reagent Water, Product Code W4502
  • JumpStart Taq DNA Polymerase (D9307)

IV. Protocol for Extraction of DNA from Soil

  • Performed with UltraClean Soil Kit by MO Bio Laboratories Inc.
    1. Add 0.5 gm of soil sample to 2 ml Bead Solution tubes provided.
    2. Gently vortex to mix.
    3. Add 60 l of Solution 1 and invert several times.
    4. Add 200 l of Solution IRS (Inhibitor Removal Solution).
    5. Vortex at maximum speed for 10 minutes.
    6. Centrifuge at 10,000 x g for 30 seconds (be sure NOT to exceed 10,000 x g or the tubes may break).
    7. Transfer the supernatant to a clean microcentrifuge tube (supernatant may contain some soil particles).
    8. Add 250 l of Solution S2 and vortex for 5 seconds.
    9. Incubate at 4 C for 5 minutes.
    10. Centrifuge the tubes at 10,000 x g for 1 minute.
    11. Transfer 450 l of supernatant to a clean microcentrifuge tube.
    12. Add 900 l of Solution S3 to the supernatant and vortex for 5 seconds.
    13. Load 700 l onto a spin filter and centrifuge at 10,000 x g for 1 minute.
    14. Discard the flow through and add the remaining supernatant to the spin filter and centrifuge at 10,000 x g for 1 minute.
    15. Add 300 l of Solution S4 and centrifuge for 30 seconds at 10,000 x g. Discard the flow- through.
    16. Centrifuge again for 1 minute.
    17. Carefully place the spin filter in a clean microcentrifuge tube.
    18. Add 50 l of Solution S5 to the filter membrane.
    19. Centrifuge for 30 seconds and discard the spin filter.
    20. Store the eluted DNA at -20 C.

V. Protocol for GenomePlex Whole Genome Amplification from Soil

  • Performed with GenomePlex Whole Genome Amplification Kit (WGA1)

    Fragmentation

    1. Prepare DNA solution of 1 ng/l from soil DNA extraction protocol described above.
    2. Add 1 l of 10X Fragmentation Buf fer to 10 l DNA (1 ng/l) in a PCR tube.
    3. Place the tube in a thermal cycler at 95 C for EXACTLY 4 minutes. Note, the incubation is time sensitive and any deviation may alter results.
    4. Immediately cool the sample on ice and centrifuge briefly.

    Library Preparation

    1. Add 2 l of 1x Library Preparation Buffer.
    2. Add 1 l of Library Stabilization Solution.
    3. Mix thoroughly and place in thermal cycler at 95 C for 2 minutes.
    4. Cool the sample on ice and centrifuge briefly.
    5. Add 1 l Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.
    6. Place sample in thermal cycler and incubate as follows:
      • 16 C for 20 minutes
      • 24 C for 20 minutes
      • 37 C for 20 minutes
      • 75 C for 5 minutes
      • 4 C hold
    7. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at - 20 C up to three days.

    Amplification

    1. Add the following reagents to the entire 15 l reaction:
      • 7.5 l 10x Amplification Master Mix
      • 47.5 l Nuclease Free Water
      • 5.0 l JumpStart Taq DNA Polymerase (12.5 units)
    2. Mix thoroughly, centrifuge briefly, and begin thermocycling:

      Initial Denaturation 95 C for 3 minutes

      Perform 14 cycles as follows:

      • Denature 95 C for 15 seconds
      • Anneal/Extend 65 C for 5 minutes
    3. After cycling is complete, maintain the reactions at 4 C or store at -20 C until ready for analysis or purification.

VI. Quantification of Amplified Products
The amount of amplified genomic DNA products can be detected with or without purification. For the highest quality samples of DNA we strongly recommend cleaning up the samples after amplification. The amplified products can be measured with the PicoGreen dsDNA Quantitation Assay (Molecular Probes Inc. Product # P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop instrument.

VII. Purification of Amplified Products

  • Performed with GenElute PCR Clean-Up Kit (NA1020)
    1. Insert a GenElute Miniprep Binding Column (with a blue o-ring) into a provided collection tube, if not already assembled. Add 0.5 ml of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the eluate.

      Note: The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.

    2. Add 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 l of Binding Solution to 100 l of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000-16,000 Xg) for 1 minute. Discard the eluate, but retain the collection tube.

    3. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1 minute. Discard the eluate, but retain the collection tube.

      Note: Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.

    4. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2 minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube.

    5. Transfer the column to a fresh 2 ml collection tube. Apply 50 l of Elution Solution or water to the center of each column. Incubate at room temperature for 1 minute.

      Note: When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using th e Elution Solution diluted 10-fold with water.

    6. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the eluate and is ready for immediate use or storage at -20 C.

Appendix

References
1. Barker, D. L., et al. Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genomic Research, 14, 901-7 (2004).

2. Gribble, S., et al. Chromosome paints from single copies of chromosomes. Chromosome Research, 12, 143-51 (2004).

3. Thorstenson, Y. R., et al. An Automated Hydrodynamic Process for Controlled, Unbiased DNA Shearing. Genome Research, 8, 848-855 (1998).

Contact Information
For technical assistance please contact:
Technical Service
(800) 325-5832
www.techserv@sial.com

To learn more about GenomePlex WGA technology visit: www.sigmaaldrich.com

GenomePlex is a registered trademark of Rubicon Genomics, Inc.
GenomePlex WGA technology patent pending.
GenElute and Jumpstart are trademarks of Sigma-Aldrich, Inc.
Nanodrop is a registered trademark of Nanodrop Technologies, Inc.
PicoGreen is a trademark of Molecular Probes, Inc.
UltraClean is a trademark of Mo Bio Laboratories, Inc.
BeadArray is a Trademark of Illumina, Inc.
TaqMan is a registered trademark of Roche Molecular Systems
Taq antibody licensed for in vitro research use under U.S. Patent No. 5,338,671 and 5,587,287, and corresponding patents in other countries.
This product is sold under license from Roche Molecular Systems, Inc. and Applied Biosystems.

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