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Samplix Xdrop Скачать руководство пользователя страница 12

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Chapter 3: dPCR Setup

Preparations for dPCR

Use the Samplix Primer test PCR kit (Cat. No. RE10200) to determine the optimal primer concentration and annealing
temp for the dPCR reaction in the Samplix dPCR mix (2X)

●

as described in the previous chapter (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.

Samplix provides a Positive control kit (Cat. No. CO10200) that contains DNA template and two primer pairs; one for
dPCR and one for validation qPCR after dMDA. See instructions next page.

Setup of dPCR reaction

1.

Prepare the dPCR reaction mix after the table below.

2.

Prepare mix for all reactions (40 μl/reaction total). Keep the reagents and the dPCR reaction mix on ice.

3.

Prepare dilutions of the correct amount of template DNA. Calculate the optimal amount of input template DNA
using the online sample calculation tool

samplix.com

4.

Add 2 μl of template DNA dilution to 38 μl dPCR reaction mix to get 40 μl/reaction total.

5.

Keep the reaction mix at 4°C until loading on dPCR cartridge followed by immediate insertion of the dPCR cartridge
on the Xdrop™ instrument.

dPCR reaction mix

1X

H

2

O (molecular grade)

17,2 μl

dPCR mix (2x)

●

20 μl

Primer Forward 10 µM

0,4 μl

Primer Reverse 10 µM

0,4 μl

Total mix

38 μl

Template DNA

2 μl

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Содержание Xdrop

Страница 1: ...Xdrop manual Droplet PCR dPCR User manual v 1 0 released 26 Aug 2019 ...

Страница 2: ...l 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 S...

Страница 3: ...luted 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 dropl...

Страница 4: ...or 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 wh...

Страница 5: ...nt 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 ...

Страница 6: ...y 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 envir...

Страница 7: ... 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 tes...

Страница 8: ...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 tube...

Страница 9: ...rget 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 comp...

Страница 10: ...e 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 ...

Страница 11: ... the best annealing temperature Use the Samplix primer test PCR kit Cat No RE10200 with Samplix dPCR mix 2X and Samplix qPCR dye to verify the primers and reaction efficiency To calculate the PCR efficiency run a qPCR with a standard curve with at least three different amounts of input DNA using the Samplix dPCR mix and your designed primer pairs Fig 2 1 Calculate the PCR efficiency using the Ct v...

Страница 12: ... and one for validation qPCR after dMDA See instructions next page Setup of dPCR reaction 1 Prepare the dPCR reaction mix after the table below 2 Prepare mix for all reactions 40 μl reaction total Keep the reagents and the dPCR reaction mix on ice 3 Prepare dilutions of the correct amount of template DNA Calculate the optimal amount of input template DNA using the online sample calculation tool sa...

Страница 13: ...al Droplet MDA dMDA Validate enrichment as described in XdropTM Manual Droplet MDA dMDA using Enrichment validation primers Set up the positive control dPCR reaction as detailed below 1 Prepare the positive control dPCR reaction mix after the table below using dPCR control primers and Positive control DNA 2 Prepare mix for all reactions 40 μl reaction total Keep the reagents and the dPCR reaction ...

Страница 14: ...ng the cartridge Hold the cartridge by its sides Do not touch any of the input wells or droplet exit well Do not touch the black microfluidic chip on the back of the cartridge Save the cartridge storage bag for later storage of the cartridge 4 Be careful not to use the same line more than once as this will disrupt droplet production To avoid using the same line more than once mark the storage plas...

Страница 15: ...8 Load 40 μl dPCR reaction mix including primers and sample DNA into well C 9 Load 100 μl dPCR oil into well B 10 Notice that the order of loading is 300 μl 1x dPCR buffer in the well A 40 μl 1x dPCR buffer to the shelf D 40 μl 1x dPCR reaction mix in well C 100 μl dPCR oil in well B 11 Add a gasket to the top of the cartridge Orient the gasket to the cartridge using the angled corner Attach to th...

Страница 16: ...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 Make sure that the cartridge is correctly positioned into the drawer Fig 3 5 as it may otherwise cause damage to the instrument Once the cartridge is correctly inserted press close to retract the drawer into the instrume...

Страница 17: ...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 Fig 3 7 Selecting the lines to be used Selected channels will be indicated by green buttons here 2 4 6 and 8 Blue buttons indicate channels not yet selec...

Страница 18: ...utton and enter an appropriate name for your log file 19 Press Open to eject the cartridge 20 Remove the cartridge from the instrument and place it in a LAF bench 21 Press Close to make the instrument retract the drawer back into its closed position 22 Press Finish to return to the Welcome screen 23 Shut down the instrument after a completed droplet production to avoid damage to the instrument If ...

Страница 19: ...oplets into four PCR tube aliquots of approximately 80 90 μl NB Please note that dPCR droplets sediment rapidly during handling To ensure equal distribution into the aliquots be sure to mix gently by pipetting up and down between each pipetting step 30 Place the PCR tubes in a thermocycler and run the following program A temperature ramp rate of 1 5 C sec or less is suggested Temperature Duration ...

Страница 20: ...an 0 02 of total dPCR droplets Therefore a positive population can be difficult to identify Make sure the live plot of fluorescence shows at least 100 000 events see fig 4 2 A histogram plot is not recommended Preparation of droplets for flow cytometry 1 Remove tubes with dPCR droplets from the PCR machine 2 Make sure the 2x dPCR buffer is diluted with molecular grade water to 1x Mix well by vorte...

Страница 21: ...s Same data on two different instruments Left Density view of all events from a flow cytometry software FCS Express 6 Right Dot plot showing dPCR droplets inside the green gate SONY SH800 Cell sorter software SONY 6 Gating the identified dPCR droplets in a new plot identify a positive and a negative green fluorescent population of droplets Using a green filter the positive fluorescent population c...

Страница 22: ...inverting the tube at least 20 times 2 Stain the Control droplets with Droplet dye as follows Prepare flow cytometry buffer by adding 500 μl 1x dPCR buffer to a flow cytometry tube tubes depend on flow cytometer instrument Spin down the vial of Droplet dye 1 2 minutes Add 5 μl Droplet dye into the flow cytometry tube with dPCR buffer Mix gently to dissolve the dye in the dPCR buffer Transfer 50 μl...

Страница 23: ...w a strict gate around the population of interest NB be aware of potential drift in fluorescence over time and be prepared to move the sorting gate during the sort if required 3 Add 15 μl of molecular grade H2O into the bottom of a 1 5 ml low binding collection tube and place the collection tube in the appropriate holder for collection in the cell sorter instrument 4 Start sorting the positive pop...

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