REF : IMG 116 - AMPLITECH Imegen... · 2017. 11. 17. · imegen Quimera Screening Multiplex Plus REF: IMG-116-26 and imegen Quimera Screening Multiplex Plus II REF: IMG-116-25 imegen

Instituto de Medicina Genómica SL

Agustín Escardino 9,

Parc Científic de la Universitat de València

46980 Paterna (Valencia, España)

+34 963 212 340 - [email protected]

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Rev. 3

Polymorphisms quantification in hematopoietic chimeras by digital PCR

REF : IMG-116






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*imegenTM

is a trademark of Instituto de Medicina Genómica, registered as

imegen® in Spain






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β






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imegen Quimera Screening Multiplex Plus REF: IMG-116-26 and imegen

Quimera Screening Multiplex Plus II

REF: IMG-116-25

imegen Quimera

(REF:Table1)






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β

INDELs Insertion

(Alelo +)

Deletion

(Alelo -) Kit reference

SRY X IMG-116-27 DRY

RhD X IMG-116-28 DRY

Q116-6I X IMG-116-32 DRY

Q116-3I X IMG-116-29 DRY

Q116-7I X IMG-116-33 DRY

Q116-12D X IMG-116-36 DRY

Q116-11I X IMG-116-34 DRY

Q116-5I X IMG-116-31 DRY

Q116-4I X IMG-116-30 DRY






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INDELs Insertion

(Alelo +)

Deletion

(Alelo -) Kit reference

Q116-10I X IMG-116-35 DRY

Q116-23I X IMG-116-37 DRY

Q116-28I X IMG-116-42 DRY

Q116-32I X IMG-116-46 DRY

Q116-31I X IMG-116-45 DRY

Q116-30D X IMG-116-44 DRY

Q116-29D X IMG-116-43 DRY

Q116-27D X IMG-116-41 DRY

Q116-24I X IMG-116-38 DRY

Q116-9I X IMG-116-48 Dry

Q116-20I X IMG-116-40 Dry

Q116-33I X IMG-116-47 Dry

Q116-37I X IMG-116-49 Dry

Q116-38I X IMG-116-50 Dry

Q116-39I X IMG-116-51 Dry

Q116-41I X IMG-116-52 Dry

Q116-42I X IMG-116-53 Dry

Q116-43I X IMG-116-54 Dry

Q116-44I X IMG-116-55 Dry

Q116-45I X IMG-116-56 Dry

Q116-46I X IMG-116-60 Dry

Q116-47I X IMG-116-57 Dry

Q116-49I X IMG-116-58 Dry

Q116-50I X IMG-116-59 Dry

Table 1. imegen-Quimera reference kit for dPCR






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Reagents Reactions Storage

Polymorphism Master Mix 2 x 6 rxn 4ºC

Table 2. imegen-Quimera kit for dPCR contents and storage temperature






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Equipment 1 Micropipettes (10 µL, 20 µL and 200 µL)

QuantStudio® 3D Digital PCR system (ThermoFisher)

2 dPCR Thermal cycler: ProFlex™ 2x Flat PCR System from ThermoFisher Scientific

3 QuantStudio 3D Digital PCR instrument (chip reader) from ThermoFisher Scientific

4 QuantStudio 3D Digital PCR Chip Loader from ThermoFisher Scientific

Droplet DigitalTM PCR system (BIO-RAD)

5 QX200TM Droplet DigitalTM PCR system o QX100TM Droplet DigitalTM PCR system

6 PX1™ PCR Plate Sealer

7 C1000 Touch™ Thermal Cycler with 96–Deep Well Reaction Module

8 Multichannel pipette (10-100 µL)

http://www.bio-rad.com/es-es/sku/1814000-px1-pcr-plate-sealer

http://www.bio-rad.com/es-es/sku/1851197-c1000-touch-thermal-cycler-with-96-ndash-deep-well-reaction-module

http://www.bio-rad.com/es-es/sku/1851197-c1000-touch-thermal-cycler-with-96-ndash-deep-well-reaction-module






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Materials 1 0.2 ml tubes

2 Disposable micropipette filter tips (10 µL, 20 µL

and 200 µL)

3 1.5 mL sterile tubes

4 Powder-free latex gloves

QuantStudio® 3D Digital PCR system (ThermoFisher)

5 QuantStudio® 3D Digital PCR 20K Chip Kit v2 (12

pack) from ThermoFisher Scientific

Droplet DigitalTM PCR system (BIO-RAD)

6 Droplet Generator Cartridges and Gaskets (Ref.1864007)

7 ddPCR 96-well plates

8 Droplet Generation Oil for probes






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Reagents Rehydration volume

Polymorphism Master Mix 10 μL of water/vial*

Table 3. Kit components rehydration volume

ᵦ






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Reagents Amount per reaction

Polymorphism Master Mix 1.5 L

Digital PCR v2 Master Mix 7.5 L

Table 4. Reagents amount required per reaction






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Reagents Amount per reaction

Polymorphism Master Mix 1.5 L

ddPCRTM Supermix for

Probes (No dUTP) 10 L

Nuclease free wáter* 5.5 L

Table 5. Reagents amount required per reaction






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Fields

Step 1

Enzymatic

activation

Step 2

PCR

Step 3

Cycles Number

1 Initial Cycle

(Denat.)

40 cycles

1 Final Cycle Conservation Primer binding/

Extension Denat.

Temperature 96ºC 56ºC 98ºC 60ºC 10ºC

Time 10 minutes 2 minutes 30

seconds 2 minutes ∞

Table 5. dPCR optimal program for imegen-Quimera Kits for dPCR

http://www.thermofisher.com/







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http://www.bio-rad.com/






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Fields

Step 1

Enzymatic

activation

Step 2

PCR

Step 3

Cycles Number

1 Initial Cycle

(Denat.)

40 cycles

1 Final Cycle Conservation

Denat.

Primer binding/

Extension

Temperature 96ºC 94ºC 60ºC 98ºC 4ºC

Time 10 minutes 30 seconds 1 minute 10 minutes ∞

Table 6. dPCR optimal program for imegen-Quimera Kits for dPCR of BIO-RAD platform







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Probe Labelling β-globin VIC®

Informative polymorphism FAMTM







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o

𝑪𝒉𝒊𝒎𝒆𝒓𝒂 % =

𝑪𝒐𝒑𝒊𝒆𝒔µ𝑳

(𝑭𝑨𝑴)


(𝑽𝑰𝑪)𝒙 𝟏𝟎𝟎

o

% 𝑸𝒖𝒊𝒎𝒆𝒓𝒂 =


(𝑭𝑨𝑴)










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o

𝑪𝒉𝒊𝒎𝒆𝒓𝒂 % =


(𝑭𝑨𝑴)



o

% 𝑸𝒖𝒊𝒎𝒆𝒓𝒂 =


(𝑭𝑨𝑴)








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https://youtu.be/K38cV3hacm8

Documents

REF : IMG 116 - AMPLITECH Imegen... · 2017. 11. 17. · imegen Quimera Screening Multiplex Plus REF: IMG-116-26 and imegen Quimera Screening Multiplex Plus II REF: IMG-116-25 imegen