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Samplix Xdrop Manual

Droplet pcr (dpcr)
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Xdrop™ manual
Droplet PCR (dPCR)
User manual v. 1.0 released 26 Aug. 2019

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Summary of Contents for Samplix Xdrop

  • Page 1 Xdrop™ manual Droplet PCR (dPCR) User manual v. 1.0 released 26 Aug. 2019...

  • Page 2: Table Of Contents

    03 Targeted enrichment overview Xdrop™ instrument overview Required items for Xdrop™ dPCR Suggested Samplix products Equipment and reagents for Xdrop™ supplied by the user Chapter 2: dPCR – General Considerations page 09 dPCR assay design Target sequence Primer design guidelines...

  • Page 3: Chapter 1: Xdrop

    Using the Xdrop™ instrument, cartridges, and reagents, sample DNA is partitioned into millions of picolitre size highly stable droplets. The Xdrop™ droplet PCR (dPCR) droplets are suitable for standard PCR cycling, flow cytometry analysis and sorting.

  • Page 4: Xdrop™ Instrument Overview

    Xdrop™ instrument overview The Xdrop™ droplet generator is compatible with Samplix dPCR cartridges for production of dPCR droplets and Samplix dMDA cartridges for the generation of droplets for amplification of DNA. When using dMDA cartridges, always use the accompanying holder. The Xdrop™ droplet generator is used for generating both dPCR and dMDA droplets.
  • Page 5 To find technical support, contact the technical support team at support@samplix.com Warranty The Xdrop™ instrument and associated accessories are covered by a standard Samplix ApS warranty. Contact your local Samplix ApS office for the details of the warranty. Safety We strongly recommend that you follow the safety specifications listed in this section and throughout this manual.
  • Page 6 Intended use and intended users The Xdrop™ instrument is intended for use by trained laboratory personnel in a clean laboratory environment for DNA sample preparation from mixed DNA samples using droplet microfluidic technology.

  • Page 7: Required Items For Xdrop™ Dpcr

    Required items for Xdrop™ dPCR Xdrop™ Instrument (Cat. No. IN00100) dPCR cartridge (Cat. No. CA10100) dPCR gasket (Cat. No. GA10100) Storage film (Cat. No. FI00100) dPCR kit (Cat. No. RE10100) dPCR kit part 1 (store at -20°C) dPCR mix (2x) ●...
  • Page 8 Equipment and reagents for Xdrop™ enrichment and amplification supplied by the user In addition to required and suggested Samplix products, the following items are also suggested. Thermal cycler Real-time PCR cycler LAF (Laminar Air Flow) hood Flow cytometry analyser / cell sorter Quantification of nucleic acids –...

  • Page 9: Dpcr Assay Design

    Target sequence The Xdrop™ technology allows targeted enrichment and amplification of a genomic region of interest without the need for long-range PCR. The target DNA of interest can contain repeat regions, GC-rich regions or other regions that are otherwise difficult to amplify. Specific primers amplifying a short fragment of 100 bp is used for capturing the region of interest.

  • Page 10: Primer Design Guidelines

    Primer design guidelines The Xdrop™ enrichment technology relies on carefully designed and highly specific PCR primer pairs. Two sets of non- overlapping PCR primer pairs are required; one set of PCR primer pairs, called the dPCR set, is responsible for creating a fluorescent signal used for target enrichment.

  • Page 11: Primer Optimization

    It is advised to run a melting curve analysis with the template DNA, dPCR primers designed and Samplix reagents to check for the presence of alternative amplicons and primer-dimers. Consider running a temperature gradient to determine the best annealing temperature.

  • Page 12: Chapter 3: Dpcr Setup

    (Chapter 2: dPCR – General Considerations). NB: Do not use any other reagents instead of Samplix dPCR kit (Cat. No. RE10100) for Xdrop™ dPCR droplet production as this may compromise droplet production, droplet stability, and downstream enrichment.
  • Page 13 The Positive control kit (Cat. No. CO10200) is designed as a positive control assay to use together with the dPCR kit (Cat. No. RE10100) and dMDA kit (Cat. No. 20100) for the full Xdrop™ workflow; dPCR droplet production, sorting and enrichment.

  • Page 14: Prepare Dpcr Cartridge

    Prepare dPCR cartridge Dilute the 2X dPCR buffer ● to 1X with molecular grade water. NB: Dilute an entire vial of dPCR buffer. Precipitate potentially forming in the 2x buffer will dissolve when diluted to 1x. Make sure to mix well by vortexing 10 seconds and inverting the tube >20 times.
  • Page 15 Fig. 3.2. Loading of the dPCR cartridge. Left: Avoid introducing air bubbles by pipetting on the side of the wells. Right: dPCR cartridge top view with wells marked. Load 300 μl 1x dPCR buffer in the first well (#A). It is important to load the cartridge in the order described here and avoid air bubbles by pipetting carefully on the side of the well (Fig.
  • Page 16 12. Push “open” on the instrument touch screen to eject the drawer (Fig. 3.4). ™ Fig. 3.4. The Xdrop instrument “welcome” screen, press “open” to eject the drawer. 13. When the “open” or “next” button has been pressed, the screen shifts to “Please place cartridge” and “close”.
  • Page 17 “dPCR” option on the touch screen (Fig. 3.6.). ™ Fig. 3.6. The Xdrop instrument “Select cartridge” screen, select dPCR cartridge. 16. The lines to be processed are selected by pressing the corresponding numbers 1-8 on the screen. When selected, buttons turn green (green = selected & blue = not selected) (Fig. 3.7).
  • Page 18 The instrument will now build up pressure. This process can take up to 4 minutes. Once the desired pressures have been built up the message “Making your droplets” and the remaining run time are displayed on the screen (Fig. 3.8). The Xdrop™ instrument will produce droplets in 40 minutes. Fig. 3.8.

  • Page 19: Collect Generated Droplets

    Collect generated droplets 24. After finalized droplet production, confirm that dPCR droplets have been produced. dPCR droplets will sink to the bottom of the collection well and form a white layer with a clear buffer phase on top. 25. If droplets are visible, collect droplets from the Exit-well (#D). 26.

  • Page 20: Chapter 4: Single Dna Molecule Detection And Sorting Of Droplets

    Chapter 4: Single DNA Molecule Detection and Sorting of Droplets Requirements for flow cytometer analyzer and cell sorter  A 488 nm (blue) laser.  Optical configuration detecting fluorophores excited at 488 nm such as FITC, GFP, and PE.  100 μm nozzle tip/sorting chip minimum.

  • Page 21: Flow Cytometry Analysis Of Dpcr Droplets

    Flow cytometry analysis of dPCR droplets Identify the dPCR droplet population on a plot of FSC (height) versus SSC (height) (Fig. 4.1). dPCR droplets are higher in side scatter (SSC) than oil droplets. Note that it could take a few minutes before the heavy dPCR droplets are analyzed and visible in the plot.
  • Page 22 Optional: Set up flow cytometry with Cell sorter control kit (Cat. No. CO10100) To easily set up flow cytometry of dPCR droplets, use the Samplix Cell sorter control kit (Cat. No. CO10100). Make sure the 2x dPCR buffer ●...

  • Page 23: Sorting Of Pcr Positive Dpcr Droplets

    DNA of interest is enriched. Depending on the input, you will get few DNA molecules after sorting, too low an amount to quantify directly. To amplify the sorted DNA, please continue with the general amplification dMDA kit developed for the Xdrop™ technology. Please refer to the Xdrop Manual Droplet MDA (dMDA).

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