Samplix Xdrop Manuel utilisateur
Xdrop™ manual
Droplet PCR (
dPCR)
User manual v. 1.0 released 26 Aug. 2019
2
Table of Contents
Chapter 1: Xdrop
TM
at a Glance page 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
DNA sample preparation
Primer optimization
Chapter 3: dPCR Setup page 12
Preparations for dPCR
Setup of dPCR reaction
Positive control dPCR reaction
Prepare dPCR cartridge
Collect generated droplets
Chapter 4: Single DNA Molecule Detection and Sorting of Droplets page 20
Requirements for flow cytometer analyzer and cell sorter
Notes to operator
Preparation of droplets for flow cytometry
Flow cytometry analysis of dPCR droplets
Set up flow cytometry with Cell sorter control droplets
Sorting of PCR positive dPCR droplets
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 [email protected]
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.
6
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 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) ●
Droplet dye ●
dPCR kit part 2 (store at -20°C)
dPCR buffer (2x) ●
dPCR kit part 3 (store at room temperature)
dPCR oil ●
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: droplet PCR – General
Considerations
dPCR assay design
The Xdrop™ technology requires a simple assay design with the following components:
A DNA sample of high purity. Calculate the required amount of input DNA needed based on the desired
enrichment and desired amount of output DNA using the online sample input calculation tool at
samplix.com
One droplet PCR (dPCR) primer pair for enrichment. Please see the design guidelines below and use the online
primer design tool at samplix.com.
One quantitative PCR (qPCR) primer pair for validation of Xdrop™ DNA enrichment. Please see the design
guidelines below and use the online primer design tool at samplix.com.
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. The Xdrop™ technology compartmentalizes the amplification reaction in small droplets and makes use of a
highly processive DNA polymerase to enable amplification of almost all regions of the genome.
The length of the enriched target DNA will depend on the length of the input DNA. Consider using high molecular
weight DNA as input with a DNA fragment size >30 kb and of high purity. Calculate the optimal amount of input
template DNA using the online sample input calculation tool at samplix.com
10
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. The second set of PCR primers, called the qPCR set, is used to validate
the assay and quantify the number of target fragments in the pool of enriched fragments.
Help for designing primers can be found in the online primer design tool at samplix.com
General design guidelines for dPCR primer pairs are as follows:
Amplicon length between 120 and 160 base pairs.
Melting temperature around 60°C.
Avoid primer pairs with more than 2°C difference in melting temperature between forward and reverse
primer.
Avoid placing primers in low complexity regions on the target.
Primers need to be specific. Avoid primer pairs that amplify sequences not related to the target sequence.
Follow the general considerations for PCR primer pairs, avoid self-complementarity, stable secondary
structures, etc.
The second primer pair required is for validation and calculation of the enrichment. NB: The second primer pair (the
qPCR primer pair) must be different from the enrichment dPCR primer pair and amplicons must not overlap.
General design guidelines for qPCR primer pairs for validation of enrichment are as follows:
Amplicon length between 80-120 base pairs.
Melting temperature around 60°C.
Avoid primer pairs with more than 2°C difference in melting temperature between the two primers.
Place the amplicon within 2 kb of the droplet primer pair without overlapping the dPCR amplicon. The risk of
false-negative validation increases if the validation qPCR assay is placed further from the dPCR assay.
Follow the general considerations for PCR primer pairs, avoid self-complementarity, stable secondary
structures, etc.
DNA sample preparation
When purifying the DNA sample, use a method that maintains the integrity of the DNA fragments while giving pure DNA
without contaminations. The Xdrop™ enrichment technology can be affected by contamination of the DNA sample by
RNA, proteins, carbohydrates, salt and phenol among others. Purify the DNA to the same quality as required for long
read sequencing.
Calculate the optimal amount of input template DNA using the online enrichment calculation tool at samplix.com
Autres manuels pour Xdrop
1
Table des matières
Autres manuels Samplix Équipement de laboratoire
Manuels Équipement de laboratoire populaires d'autres marques
Agilent Technologies
Agilent Technologies 5800 ICP-OES Manuel utilisateur
Endress+Hauser
Endress+Hauser Cleanfit CPA875 Manuel utilisateur
NI
NI PXI-5422 Manuel
Collomix
Collomix Aqix Manuel utilisateur
SPEX SamplePrep
SPEX SamplePrep 6875 Freezer/Mill Series Manuel utilisateur
Ocean Insight
Ocean Insight FLAME-NIR+ Manuel utilisateur
