Navigation Links
Protocol for GenomePlex Whole Genome Amplification from Blood Card

Application Guide

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

Application Guide

I. Description
Blood cards provide the convenience of archiving small volumes of blood. Many times genomic DNA from samples can have low yield. This quantity can hinder the researcher's ability to perform downstream analysis. This method describes amplifying nanogram amounts of starting genomic DNA with little or no detectable bias resulting in microgram quantities. In addition, the protocol provides a simple and convenient method to extract the genomic DNA from the blood card, amplify the genomic DNA, and rapidly purify the PCR amplified products. The methods described are completed using the GenElute Blood Genomic DNA Kit, GenomePlex WGA kit, and GenElute PCR Clean-up Kit.

GenomePlex is a Whole Genome Amplification (WGA) method that allows the researcher to generate a representative, approximate 300 to 1000-fold amplification of genomic DNA. The amplification yield is dependent on the purity and amount of starting material. The kit utilizes a proprietary amplification technology based upon random fragmentation of genomic DNA and conversion of the resulting small fragments to PCR-amplifiable OmniPlex Library molecules flanked by universal priming sites. The OmniPlex library is then PCR amplified using universal oligonucleotide primers and a limited number of cycles. This technology maintains the genetic representation with concordance >99.8% in genotyping results from comparing genomic DNA and GenomePlex amplified DNA1. In addition to SNP genotyping, downstream applications also include performing TaqMan assays and BeadArray analysis.

II. Product Components

  • GenElute Blood Genomic DNA Kit (NA2000)
  • GenomePlex Whole Genome Amplification Kit (WGA1)
  • GenElute PCR Clean-Up Kit (NA1020)
III. Materials to be Supplied by the User
  • Blood Card
  • 1.5 ml microcentrifuge tubes for lysis
  • Microcentrifuge (with rotor for 2ml tubes)
  • Ethanol (absolute) Product Code E7023
  • Molecular Biology Reagent Water, Product Code W4502
  • JumpStart Taq DNA Polymerase, Product Code D9307
  • 55 C water bath or heating block
IV. Protocol for Extraction of DNA from Blood Card
  • Performed with GenElute Blood Genomic DNA (NA2000)
    1. Cut a disc from a dried blood card (200 l spotted) into several 2 mm by 2 mm pieces and place the pieces into a 1.5 ml microcentrifuge tube.
    2. Add 40 l Proteinase K and 1.0 ml Resuspension Solution.
    3. Add 300 l of Lysis Solution C and vortex thoroughly for 15 seconds.
    4. Incubate at 55 C for 10 minutes.
    5. After the incubation, transfer the liquid (discard blood card remaining in the microcentrifuge tube) to a 15ml conical tube.
    6. Add 500 l of Column Preparation Solution to the GenElute Miniprep Binding Column (red o-ring) and centrifuge at 12,000 x g for 1 minute. Discard the flow-through liquid.
    7. Add 900 l of 95-100% ethanol to the lysate in the 15 ml conical tube and mix thoroughly by vortexing 5 to 10 seconds.
    8. Transfer the contents of the tube into the treated column from step 4. Centrifuge at ≥ 6,500 x g for 1 minute. Repeat this until all of the lysate has been passed through the column.
    9. Discard the collection tube and flow-through and place the column in a new 2 ml collection tube.
    10. Add 500 l of Prewash Solution (be sure to dilute with ethanol prior to first use) and centrifuge for 1 minute at ≥ 6,500 x g for 1 minute.
    11. Discard the collection tube containing the flow through and place the binding column in a new 2 ml collection tube.
    12. Add 500 l of Wash Solution (be sure to dilute with ethanol prior to first use) to the binding column and centrifuge at maximum speed (12,000 x g to 16,000 x g) for 3 minutes to dry the binding column.
    13. Pipette 400 l of Elution Solution onto the column and centrifuge for 1 minute at ≥ 6,500 x g to elute the DNA.
    14. Store the eluted DNA at -20 C.
V. Protocol for GenomePlex Whole Genome Amplification from Blood Card
  • Performed with GenomePlex Whole Genome Amplification Kit (WGA1)

    Fragmentation

    1. Prepare DNA solution of 1 ng/l from whole blood extraction protocol described above.
    2. Add 1 l of 10X Fragmentation Buffer 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 GenomePlex Whole Genome Amplification Kit 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 the 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

Application Data

GenomePlex WGA human genomic DNA from Blood Card

Lane 1 - 1kb Marker
Lane 2 - Sigma Positive Control
Lane 3 - Sigma Negative Control
Lane 4 - Sigma Blood Card
Lane 5 - Sigma Blood Card
Lane 6 - Competitor A Positive Control
Lane 7 - Competitor A Negative Control
Lane 8 - Competitor A Blood Card
Lane 9 - Competitor A Blood Card
Lane 10 - Competitor Q Positive Control
Lane 11 - Competitor Q Negative Control
Lane 12 - Competitor Q Blood Card
Lane 13 - Competitor Q Blood Card
Lane 14 - 1kb Marker

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.
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.

'"/>

Source:


Page: All 1 2 3 4 5 6

Related biology technology :

1. New Protocols for Isolating High- Molecular-Weight Genomic DNA
2. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
3. Lambda DNA Extraction Protocol
4. M13/Phagemid DNA Extraction Protocol
5. Mouse Tail Genomic DNA Isolation Protocol(1)
6. Genomic DNA Isolation Protocol(1,2,3)
7. Basic Plasmid DNA Isolation Protocol
8. Protocol for RNA Isolation Using TRIzol Reagent with Phase Lock Gel-Heavy
9. RNA-free Plasmid DNA Isolation Protocol
10. Lambda DNA Extraction Protocol
11. Eppendorf Multiporator Transfection Protocols for Eukaryotic Cells
Post Your Comments:
(Date:9/2/2014)... 2, 2014 Orexigen Therapeutics, Inc. (Nasdaq: ... and Trademark Office (PTO) has issued a patent related ... weight loss. NB32 is a fixed-dose combination of naltrexone ... Patent No. 8,815,889 claims methods for treating insulin resistance ... expires in 2024. If NB32 is approved for use ...
(Date:9/2/2014)... ROCKVILLE, Md. , Sept. 2, 2014 /PRNewswire/ ... of novel anti-infective biologic and drug candidates targeting ... announced today positive results from its final preclinical ... Drug Administration (FDA) guidance, this bridging study was ... product candidate designed to prevent the devastating effects ...
(Date:9/2/2014)... BETHESDA, Md. , Sept. 2, 2014  Spherix ... fostering and monetization of intellectual property, today announced that ... three patents to the Company in the month of ... of patents.  The issued patents are: ... , U.S.RE45,081 issued August 19, 2014; and ...
(Date:9/1/2014)... , Sept. 1, 2014 Reportlinker.com ... available in its catalogue: India Solid ... http://www.reportlinker.com/p02350445/India-Solid-Waste-Management-Vehicles-Market-Forecast-and-Opportunities-2019.html ... are the main governing bodies responsible for complete ... cities. On an average, around 135,000 MT solid ...
Breaking Biology Technology:Orexigen Expands NB32 Intellectual Property Portfolio With New U.S. Patent 2Orexigen Expands NB32 Intellectual Property Portfolio With New U.S. Patent 3Orexigen Expands NB32 Intellectual Property Portfolio With New U.S. Patent 4Synthetic Biologics to Initiate Clinical Trials of SYN-004 in 4Q 2014 to Prevent Potentially Deadly C. difficile Infections 2Synthetic Biologics to Initiate Clinical Trials of SYN-004 in 4Q 2014 to Prevent Potentially Deadly C. difficile Infections 3Synthetic Biologics to Initiate Clinical Trials of SYN-004 in 4Q 2014 to Prevent Potentially Deadly C. difficile Infections 4Synthetic Biologics to Initiate Clinical Trials of SYN-004 in 4Q 2014 to Prevent Potentially Deadly C. difficile Infections 5United States Patent & Trademark Office Issues Three New Standard Essential Patents to Spherix 2India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 2India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 3India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 4India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 5India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 6India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 7India Solid Waste Management Vehicles Market Forecast and Opportunities, 2019 8
... Switzerland, October 5, 2011 Debiopharm Group™ ... on drug development and companion diagnostics, today presented the ... Cancer Center Research Institute in Tokyo, for his basic ... genes, and to Doctor Mineo Kurokawa from the University ...
... 4, 2011 The VENTANA anti-Helicobacter pylori (SP48) Rabbit ... H. pylori antibody to receive 510(k) ... by Ventana Medical Systems, Inc. (Ventana), a member of ... used in immunohistochemical (IHC) staining, aids in the detection ...
... JACKSONVILLE, Fla., Oct. 4, 2011 MSC Care ... and services to post-discharge and post-injury workers, compensation ... Integrated Healthcare Services, a national company also focused ... unique and comprehensive surgical implant cost management solution, ...
Cached Biology Technology:The Japanese Cancer Association and Debiopharm Group™ Present Doctors Arakawa and Kurokawa With the 2011 'JCA-Mauvernay Award' for Their Innovative and Outstanding Research in the Field of Cancer 2The Japanese Cancer Association and Debiopharm Group™ Present Doctors Arakawa and Kurokawa With the 2011 'JCA-Mauvernay Award' for Their Innovative and Outstanding Research in the Field of Cancer 3Ventana Medical Systems, Inc. Receives First FDA 510(k) Clearance for H. pylori Antibody 2Ventana Medical Systems, Inc. Receives First FDA 510(k) Clearance for H. pylori Antibody 3MSC to Offer Surgical Implant Cost Management Solution through Acquisition of Integrated Healthcare Services 2
(Date:9/2/2014)... From AGU,s blogs: Earthquake rupture through a U.S. suburb ... University of California Davis in the hours and days ... helping scientists understand why the earthquake caused so much ... The Trembling Earth blog, hosted by the American Geophysical ... Future Mars Rovers: The Next Places to Direct Our ...
(Date:9/2/2014)... September 3, The Field Museum will present the prestigious ... of his commitment to biodiversity conservation awareness. The Parker/Gentry ... an outstanding individual, team or organization whose efforts have ... natural heritage and whose actions can serve as a ... of Mongabay.com, an environmental science and conservation news website ...
(Date:9/2/2014)... Konservat-Lagersttte of lithographic limestone is well known as ... from that area (for example, Archaeopteryx). Now, for ... in the French equivalent of these outcrops - ... oldest known water treader. , Despite the abundance ... fossils have been obtained from the Late Kimmeridgian ...
Breaking Biology News(10 mins):This week From AGU: California earthquake, future Mars rovers, models underestimate ozone 2Exceptionally well preserved insect fossils from the Rhône Valley 2
... to support the theory that old cells help make ... that as these animals age, the number of aging ... cells lose their ability to divide, a state known ... advanced online edition of Science, is the first to ...
... the journal Genome Research that large segments ... mobile DNA elements called transposons. The locations of ... are enriched in genes crucial for the regulation of ... DNA sequences that have the capacity to move from ...
... SIDS (sudden infant death syndrome) in African Americans can be ... deaths result from a common genetic variation that increases an ... of environmental stress, a research team based at the University ... Journal of Clinical Investigation. , Children with two copies ...
Cached Biology News:Aging cells, aging body: Fresh evidence for a connection 2Where 'jumping genes' fear to tread 2Gene variation increases SIDS risk in African Americans 2Gene variation increases SIDS risk in African Americans 3