Samplix Xdrop Manual de usuario
Xdrop™ manual
Droplet MDA (dMDA)
User manual v. 1.0 released 26 Aug. 2019
2
Table of Contents
Chapter 1: Xdrop™ at a Glance page 3
Targeted enrichment overview
Xdrop™ instrument overview
Required items for Xdrop™ dMDA
Suggested Samplix products
Equipment and reagents for Xdrop™ supplied by the user
Chapter 2: General DNA Amplification by dMDA page 9
Break sorted droplets
Set up dMDA reaction
Load the dMDA cartridge
Generate dMDA droplets on the Xdrop™ instrument
Chapter 3: Evaluation of Targeted Enrichment page 19
Quantify total DNA
Evaluate the enrichment of target DNA
Calculate fold enrichment of target DNA
Contact:
Samplix ApS
Mileparken 28
DK – 2730 Herlev
www.samplix.com
Mail: [email protected]
For research use only, not for use in diagnostic procedures.
Copyright 2019. Reproduction in any form, either print or electronic,
is prohibited without written permission of Samplix ApS
3
Chapter 1: Xdrop™ at a Glance
Targeted enrichment overview
Congratulations with your new Xdrop™ instrument, which we expect will facilitate groundbreaking research.
The Xdrop™ technology introduces a new approach for genomic analysis as the technology enables targeted
enrichment of genomic regions in droplets. The Xdrop™ technology offers sensitive and unbiased PCR-free sample
enrichment and general amplification prior to downstream analysis e.g. next generation sequencing.
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.
In the first step of enrichment, the sample is diluted and partitioned into millions of double emulsion droplets using the
Xdrop™ instrument and the advanced microfluidics dPCR cartridge. Droplets containing the target DNA molecules are
identified by a 120-160 base pair targeted PCR specific to a sequence within or adjacent to the region of interest.
Positive droplets are identified by their fluorescence and physically separated from negative droplets by use of a
standard cell sorter. The result is an enrichment of long single molecules comprising tens of kilobases of DNA
information.
For downstream universal amplification of the single molecules, Samplix has developed a proprietary technology by
which the high molecular weight DNA molecules are partitioned into thousands of droplets for high fidelity multiple
displacement amplification in droplets (dMDA).
The Xdrop™ enrichment and amplification technology are compatible with downstream molecular biology techniques
such as short and long read DNA sequencing.
4
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.
The Xdrop
™
droplet generator is composed of the following parts (see figure below):
Drawer – holds the dPCR or dMDA cartridge.
Touch screen – provides the means to control the droplet generator with gloved or un-gloved
hands.
USB port on the back of the instrument – connects to a USB flash drive for troubleshooting, saving log files,
and for updating instrument firmware.
Status LED – Green when in standby and operating and yellow-green when opening and closing the
drawer.
Air vents on the back of the instrument – for ventilation.
A start button on the front of the instrument.
A hardware switch on the back of the instrument.
5
Specifications
Width: 25 cm / 9,8 inches
Height: 25 cm / 9,8 inches
Length: 48 cm / 18,9 inches
Weight 17 kg / 37,5 lbs.
Voltage requirements: 110 V-240 V
Support
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.
Xdrop™ is produced to comply with Safety Requirements for Electrical Equipment for Measurement, Control, and
Laboratory Use (UL 61010-1) and complies with EU (CISPR 11, class A, group 1, 150 kHz – 30 MHz) EMC.
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Instrument safety warnings
The following warning labels refer directly to the safe use of the droplet generator.
Icon
Meaning
Warning about the risk of harm to body or equipment. Operating the Xdrop™
before reading this manual can constitute a personal injury hazard. Only
qualified laboratory personnel should operate this instrument.
Warning about the risk of harm to body or equipment from electrical shock.
Do not attempt to repair or remove the outer case of this instrument, power
supply, or other accessories. If you open these instruments, you put yourself
at risk for electrical shock and void your warranty. All repairs must be done
by an authorized repair service.
Never remove the outer case of an Xdrop
™
instrument. This may cause
you to receive an electrical shock.
Warning about the risk of harm to hands and fingers. Always keep hands
and fingers away from the instrument when the drawer is in motion.
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.
Transportation and storage
Always transport the instrument in the original Samplix box. Before starting up the instrument, let it stay in room
temperature for at least 2 hours.
Maintenance and cleaning
If the instrument is shipped back to Samplix for maintenance, please make sure that the outer surfaces are cleaned
using a cloth and 70 % ethanol.
7
Required items for dMDA
Xdrop™ Instrument (Cat. No. IN00100)
dMDA cartridge (Cat. No. CA20100)
dMDA holder (Cat. No. HO10100)
dMDA gasket (Cat. No. GA20100)
Storage films (Cat. No. FI00100)
dMDA kit (Cat. No. RE20300)
dMDA kit part 1 (store at -20°C)
dMDA-mix (5x) ●
dMDA enzyme ○
dMDA kit part 2 (store at room temperature)
dMDA oil ●
Break solution ●
Break colour ●
Suggested Samplix products
Cell sorter control kit (Cat. No. CO10100)
Cell sorter control kit part 1 (store at -20°C)
Droplet dye ●
dPCR buffer (2x) ●
Cell sorter control kit part 2 (store at 4°C)
Control droplets ○
Positive control primer kit (store at -20°C) (Cat. No. CO10200)
dPCR control primers ●
Enrichment validation primers ●
Positive control DNA ●
Primer test PCR kit (store at -20°C) (Cat. No. RE10200)
dPCR mix (2X) ●
qPCR dye (20x) ●
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 – Nanodrop, Qubit, Quantus, Bioanalyzer, Tapestation or similar
Microcentrifuge
Vortex
Freezing blocks for both PCR tubes and microcentrifuge tubes
Nuclease-free water
Nuclease-free tubes and filter pipette tips
Wide bore pipette tips (P200 Pipette, Orifice size: 1.5 mm)
PCR tubes
1.5 ml LoBind tubes
9
Chapter 2: General DNA Amplification by
dMDA
Break sorted droplets (if applicable)
If continuing the enrichment of selected DNA molecules from Xdrop™ dPCR droplets, release the DNA from the sorted
dPCR droplets by breaking the droplets with Break solution ● and Break colour ● as described below. Do not store
sorted dPCR droplets longer than a few hours as this may lead to degradation of DNA. If amplifying already purified
DNA, continue directly to “Set up dMDA reaction” page 10. For advice on the dPCR reaction see Xdrop™ manual droplet
PCR (dPCR).
1. Add 20 μl Break solution ● to each tube of sorted dPCR droplets.
2. Add 1 μl of Break colour ●. This will colour the water phase. NB: The water phase may be a colour ranging from
yellow to purple as the Break colour functions as a pH indicator.
3. Flick tube gently, do not vortex.
4. Spin tube briefly (15-30 sec).
5. Remove the clear Break solution phase from the bottom of the tube and discard. Be careful to remove all the
Break solution.
6. Repeat steps 3-5 to remove all leftover Break solution. It is important to remove all the Break solution phase as
residual Break solution may inhibit downstream enzymatic reactions.
7. Keep the coloured water phase, which will contain DNA from the dPCR droplets (Fig. 2.1).
Fig. 2.1. Break sorted dPCR droplets with Break solution
●
and Break colour
●.
Discard the clear break solution
phase at the bottom of the tube. Keep the top coloured water phase, this phase will contain your DNA molecules.
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Set up dMDA reaction
Note: Do not use any other reagents than Samplix dMDA kit (Cat. No. RE20300) for Xdrop™ dMDA droplet production
as this may compromise droplet production, droplet stability, and downstream enrichment.
Prepare amplification mix following the table below in a Laminar Air Flow (LAF) bench or similar dust free environment.
Be careful to avoid DNA contaminations of any kind.
Amplification mix
1X
H2O (molecular grade) 5 μl
dMDA mix (5x) ● 4 μl
dMDA enzyme ○ 1 μl
Template DNA solution 10 μl
Total 20 μl
1. Mix reagents as above, mix gently, no vortexing.
2. Aliquot mix in cooling block. Important! Keep cold at 4°C until use.
3. Add 10 μl template (sorted dPCR droplets after break).
4. Include the following control reaction:
10 μl molecular grade H2O, (negative control)
5. Consider preparing these suggested controls:
10 μl sheath fluid from flow cytometer, (contamination control)
10 µl 1 pg total non-sorted input DNA, (positive control)
6. Mix gently and keep cold at 4°C until loading the sample mix on the dMDA cartridge.
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