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The PicoPLEX WGA Kit, developed and manufactured by Rubicon Genomics, Inc., 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 PicoPLEX 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 ).
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 PicoPLEX 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 1XTE (pH 8.0) for storage at 4°C for up to 1 month.
Storage: Store the PicoPLEX WGA Kit at -20°C for up to 12 months from the assay date. The expiration date can be found on the box label.
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.
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.
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.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
Licenses
FOR RESEARCH USE ONLY: Developed and manufactured by Rubicon Genomics, Inc., Ann Arbor, MI, USA. Protected under US Pat. No. 8,206,913. Other US and foreign patents pending. PICOPLEX, RUBICON GENOMICS + stylized DNA design are trademarks or registered trademarks of Rubicon Genomics, Inc., used under license.
Limited Use Label License: This product and its use is the subject of one or more issued and/or pending U.S. and foreign patents and patent applications owned by Rubicon Genomics, Inc. The purchase of this product from NEB or its authorized distributors conveys to the buyer the non-transferable right to use the purchased amount of the product for Internal Research Use Only. For use of this product for Commercial, including resale of the product or use as part of a paid-for service, or diagnostic purposes, contact NEB at busdev@neb.com.
Developed and manufactured by Rubicon Genomics, Inc. Distributed by New England Biolabs, Inc.
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