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  • Single Cell WGA Kit

    Description

    The Single Cell WGA Kit efficiently amplifies total DNA from single cells or their DNA equivalent about 1 million-fold to produce 2–5 micrograms of amplified DNA in under 3 hours. The kit can also be used successfully in situations where the low input amount is uncertain as it works well over a range of inputs up to thousands of cells or several ng DNA to achieve this final yield of amplified DNA. Both AT- and GC-rich regions are represented reproducibly using this kit. With single blastomere and polar bodies, the kit is able to achieve 95% amplification success rate. The kit performs reproducibly and generates greater than 90% correlation coefficients for qPCR Ct data from replicate single-cell reactions. This method features single-copy sensitivity as well as high specificity with an expected 5 PCR cycle delay between experimental samples and non-template negative controls. The Single Cell WGA Kit produces amplified DNA fragments suitable for Copy Number Variation (CNV) analysis using oligonucleotide aCGH or qPCR; SNP genotyping, mutation detection and sequencing.

    Figure 1:
    Example of Background Subtracted RFU amplification curves for replicate single-cell and control no cell WGA reactions that were monitored on a Bio-Rad I Cycler iQ. Data provided by Rubicon Genomics, Inc.
    Figure 2:
    Workflow overview for a single cell or 15 pg-15 ng DNA (for detailed instructions please refer to protocols)
    Figure 3: Gel image of reaction products.
    The size range of amplified product from a sorted single cell or 15 pg DNA (diluted human genomic DNA equivalent to two diploid human cells). 10 μl of 75 μl reactions were analyzed by 1.4% agarose gel electrophoresis: Lane 1: no cell/DNA negative control, Lanes 2 and 3: independent reactions using 15 pg human genomic DNA. Note: The appearance of amplified product will be identical whether the starting material is a single cell or this amount of DNA. Marker M is 1 μg 2-Log DNA Ladder (NEB #N3200 ). 
    Kit Components:   #E2620S  #E2620L 
    Cell Extraction Buffer 0.1 ml  0.3 ml 
    Extraction Enzyme Dilution Buffer 0.1 ml  0.3 ml 
    Cell Extraction Enzyme 0.005 ml  0.015 ml 
    Pre-Amp Reaction Mix  0.1 ml  0.275 ml 
    Pre-Amp Enzyme 0.005 ml  0.015 ml 
    Amplification Reaction Mix 0.35 ml  1.4 ml 
    Amplification Enzyme  0.012 ml  0.05 ml 
    Nuclease-Free Water 1.8 ml  1.8 ml 

    Advantages and Features

    Features

    • Single-copy specificity
    • Low background
    • Reproducible

    Properties and Usage

    Materials Required but not Supplied

    PCR thermal cycler (Recommended: Real-time qPCR instrument)
    PCR tubes or 96-well plates for cell extraction
    PCR plate seals for pre-amp steps (Recommended: Microseal® 'F' Foil film, Bio-Rad Cat. #MSF-1001 or Axymat silicone sealing mats, Axygen Cat. #AM-96-PCR-RD)
    PCR optically clear plate sealing film for amplification steps (Recommended: Microseal "B" film, Bio-Rad Cat. #MSB-1001)
    Low Binding Barrier Filter pipette tips
    Note: Considerable (> 5 μl) evaporation may occur during step 6 of the Pre-Amplification Protocol if the incubation is being performed in a PCR tube or plate without a tight seal. Evaporation may reduce the robustness and reproducibility of the Single Cell WGA Kit. A mock step 6 incubation from the Pre-Amplification Protocol using 15 μl of water is recommended to confirm the suitability of the selected tube or plate/seal combination.
    PBS buffer for cell washes (Recommended: 20X PBS, Cell Signaling Technology Inc. Cat. #9808 or 10X PBS, USB Corporation Cat. #75889)

    Additional Optional Materials Not Included:

    DNA purification system (Recommended: DNA Clean & Concentrator™-5 Kit, Zymo Research Cat. #D4014 or MultiScreen PCR96 Plate, Millipore Cat. #MSNU03050 or QIAquick PCR Purification Kit, Qiagen #28104)
    SYBR Green I dye (Recommended: Invitrogen Cat. #S7563)
    Note: Dye can be diluted to an intermediate concentration in DMSO for storage at -20°C. For the final working stock, this intermediate stock can be further diluted in 1X TE (pH 8.0) for storage at 4°C for up to 1 month.

    Notes

    1. Storage:
      Store the Single Cell WGA Kit at -20°C for up to 12 months from the assay date. The expiration date can be found on the box label.
    2. Transfer the Cell Extraction Enzyme, Pre-Amp Enzyme and Amplification Enzyme tubes to ice just before use. All other components can be thawed on ice and briefly vortexed prior to use.
    3. This kit is designed to amplify picogram quantities of DNA, therefore extreme caution must be exercised to prevent introduction of exogenous DNA. Good laboratory practices for PCR should be followed when storing and handling reagents as well as when setting up reactions.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Manuals

    The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].
    1. How many cells can be amplified by the Single Cell WGA Kit?
    2. Does the Single Cell WGA Kit amplify genomic loci?
    3. Are all genomic loci equally represented in products amplified from the Single Cell WGA Kit?
    4. Does the Single Cell WGA Kit amplify GC-rich regions?
    5. Can negative control reactions not containing a cell or DNA be distinguished from single cell reactions?
    6. How robust is the WGA process?
    7. How rapid is the Single Cell WGA process?
    8. How long are the expected amplicons generated with the Single Cell WGA Kit?
    9. Should cells be washed before collections?
    10. Which cell collection methods are compatible with the Single Cell WGA Kit?
    11. Are there special requirements for flow sorting?
    12. What cells types have been successfully amplified by the Single Cell WGA Kit?
    13. Can the Single Cell WGA Kit be used with subcellular materials?
    14. Can the Single Cell WGA Kit be used with bacterial cells?
    1. Sample Preparation Methods for Single Cell WGA Kit
    2. Pre-Amplification Protocol for Single Cell WGA Kit
    3. Amplification Protocol for Single Cell WGA Kit