Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: VetMAX BVDV 4ALL Kit

Type: Operating instructions

For Laboratory Use.

VetMAX™BVDV4ALLKit

SUPPLEMENTAL INSTRUCTIONS

Bovine Viral Diarrhea Virus viral RNA puriﬁcation protocols

compatible with the VetMAX™BVDV4ALLKit

for use with:

Species: Bovine and small ruminants (sheep, goats)

Matrices: Blood, serum, and ear biopsies

Test type: Individual or pooled (according to the type of specimen)

Catalog NumberBVD4ALL

Document Part Number100021235

Publication NumberMAN0008962

Revision G.0

Laboratoire Service International (LSI) | 6 Allée des Ecureuils – Parc Tertiaire du Bois-Dieu | 69380 Lissieu – France

For descriptions of symbols on product labels or product documents, go to thermoﬁsher.com/symbols-deﬁnition.

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No.MAN0008962

Revision Date Description

G.0 28 September 2022

•A new chapter for liquid and wet ear biopsies was created (see Chapter 4, “RNA extraction from

liquid and wet ear biopsies”).

•The important note for ocial testing incubation time for wet ear biopsies was added (see “Prepare

the ear biopsy lysate” on page46).

•The protocols for the MagVet™ Universal Isolation Kit were removed from the document.

•Minor wording updates were completed for consistency with related documents.

F.0 30 January 2020

•Added the One‑Step Heating Workﬂow that only requires a single heating step for the rapid lysis of

ear biopsies (see Chapter5, “Rapid lysis for ear biopsies”).

•Updated the list of required materials that are not supplied with the kit.

E.0 7 November 2018

•Added instructions for nucleic acid puriﬁcation with the MagMAX™ CORE Nucleic Acid Puriﬁcation

Kit.

•Added Appendix A, “Blood storage guidelines applicable to France”.

D.0 20 June 2017 Updated to the current document template, with associated updates to the warranty, trademarks, and

logos.

C.0 16 January 2015

•Corrected NucleoSpin™ RNA kit name.

•In step 10 of “Extraction with the QIAamp™ Viral RNA Mini Kit”, added instructions to place the

column in a new collection tube.

B.0 22 April 2014 Added instructions for use of 5 - IPC BVDV 4ALL.

A.0 13 December 2013 New document.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these

products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. Eppendorf™

is a trademark of Eppendorf AG. NucleoSpin™ is a trademark of MACHEREY‑NAGEL. QIAGEN™, QIAamp™, and RNeasy™ are

trademarks of QIAGEN GmbH.

Contents

■CHAPTER1Overview .............................................................. 6

Sampleselection ................................................................ 6

Cell-free samples and wholeblood ............................................ 6

Cellsamples ............................................................... 7

Sample storage ................................................................. 7

Whole blood (in EDTAtubes) ................................................. 7

Serum ..................................................................... 7

Earbiopsies ................................................................ 7

Required materials notsupplied ................................................... 8

Recommended RNA extraction protocols ......................................... 10

■CHAPTER2RNA extraction from EDTA blood,serum ......................... 11

Preparation of samples before extraction .......................................... 11

Whole blood collected in EDTAtubes ........................................ 11

Serum for individualanalysis ................................................ 11

Pooled serum or whole bloodanalyses ....................................... 11

Guidelines for exogenous Internal Positive Control (IPC)use ......................... 12

Extraction with the MagMAX™ CORE Nucleic Acid PuriﬁcationKit .................... 12

Recommendedworkﬂows .................................................. 12

Workﬂow: Multisample Simple .............................................. 13

Workﬂow: Simple .......................................................... 13

Proceduralguidelines ...................................................... 14

Before ﬁrst use of thekit .................................................... 14

Set up the processing plates (KingFisher™ Flex and MagMAX™

Express-96instruments) .................................................. 15

Prepare samples for processing using the Multisample SimpleWorkﬂow ......... 16

Prepare samples for processing using the SimpleWorkﬂow ..................... 17

Set up the processing plates or tube strips (KingFisher™ Duo Prime and

KingFisher™mLinstruments) .............................................. 19

Process samples on theinstrument .......................................... 19

Extraction with the MagMAX™‑96 Viral RNA IsolationKit ............................ 20

Notes .................................................................... 20

Before starting ............................................................ 20

Protocol .................................................................. 21

VetMAX™BVDV4ALLKitSupplemental Instructions 3

Extraction with the QIAamp™ Viral RNA MiniKit .................................... 22

Before starting ............................................................ 22

Protocol .................................................................. 22

Extraction with the NucleoSpin™ RNA Viruskit ..................................... 23

Before starting ............................................................ 23

Protocol .................................................................. 24

■CHAPTER3RNA extraction from dry earbiopsies ............................. 26

Extraction with the MagMAX™ CORE Nucleic Acid PuriﬁcationKit .................... 27

Workﬂow ................................................................. 27

Proceduralguidelines ...................................................... 28

Guidelines for sample storage ............................................... 29

Before ﬁrst use of thekit .................................................... 29

Set up the processing plates (KingFisher™ Flex and MagMAX™

Express-96instruments) .................................................. 30

Prepare Lysis/Binding/ BeadMix ............................................. 30

Prepare the ear biopsy lysate ................................................ 30

Combine the lysate with PK (optional), then add Lysis/Binding/ BeadMix ......... 31

Set up the processing plates or tube strips (KingFisher™ Duo Prime and

KingFisher™mLinstruments) .............................................. 33

Process samples on theinstrument .......................................... 33

Preparation of samples before extraction with otherkits ............................. 34

Earbiopsies ............................................................... 34

Extraction with the MagMAX™-96 Viral RNA IsolationKit ............................ 34

Before starting ............................................................ 34

Protocol .................................................................. 35

Extraction with the RNeasy™ MiniKit ............................................. 36

Before starting ............................................................ 36

Protocol .................................................................. 37

Extraction with the NucleoSpin™ RNAkit .......................................... 38

Before starting ............................................................ 38

Protocol .................................................................. 38

■CHAPTER4RNA extraction from liquid and wet earbiopsies ................ 40

Extraction with the MagMAX™ CORE Nucleic Acid PuriﬁcationKit .................... 40

Recommended workﬂows andscripts ........................................ 40

Multisample Simple Workﬂow: Liquid earbiopsy ............................... 41

Express Workﬂow: Wet earbiopsy ........................................... 43

Proceduralguidelines ...................................................... 43

Before ﬁrst use of thekit .................................................... 44

Prepare samples for processing using the Multisample SimpleWorkﬂow ......... 45

Prepare samples for processing using the ExpressWorkﬂow .................... 49

Contents

4VetMAX™BVDV4ALLKitSupplemental Instructions

■CHAPTER5Rapid lysis for earbiopsies ........................................ 53

Preparation of samples before extraction .......................................... 53

Earbiopsies ............................................................... 53

Rapid lysis with the VetMAX™ Ear Notch Fast LysisKit .............................. 53

Recommendedworkﬂows .................................................. 53

Before youbegin .......................................................... 53

Prepare Fast Lysis Solution(FLS) ............................................ 53

Lyse samples using the One-Step HeatingWorkﬂow ........................... 54

Lyse samples using the Two-Step HeatingWorkﬂow ........................... 54

Analyze thesamples ....................................................... 55

■APPENDIXABlood storage guidelines applicable to France ................. 56

■Documentation and support ....................................................... 57

Customer and technical support ................................................. 57

Limited product warranty ........................................................ 57

Contents

VetMAX™BVDV4ALLKitSupplemental Instructions 5

Overview

IMPORTANT! In France, follow Appendix A, “Blood storage guidelines applicable to France”.

Species Isolation of nucleic acid from

matrices Test type

Bovine

Small ruminants (sheep, goats)

Blood

Serum

Ear biopsies

Individual or pooled

(according to the type of specimen)

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear

appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from

thermoﬁsher.com/support.

WARNING! POTENTIAL BIOHAZARD. Read the biological hazard safety information at this

product's page at thermoﬁsher.com. Wear appropriate protective eyewear, clothing, and gloves.

Sample selection

Cell-free samples and whole blood

Animal Sample matrix Type of analysis Sampling equipment

Calf less than 1 month old Whole blood Individual[1] EDTA tube

Serum Individual[1] Dry tube with or without separator

Cattle more than 1 month old Whole blood Individual or pooled EDTA tube

Serum Individual or pooled Dry tube with or without separator

[1] It is recommended to test this sample type individually.

Refer to the national, regional, or local regulations in eect for the size of the pools.

6VetMAX™BVDV4ALLKitSupplemental Instructions

Cell samples

Animal Sample matrix Type of analysis Sampling equipment

Calves Skin samples Individual or pooled Ear biopsies

Refer to the national, regional, or local regulations in eect for the size of the pools.

Sample storage

The quality of the samples must be assured before starting the analytical process.

Whole blood (in EDTA tubes)

Collect blood in EDTA tubes. Following collection, maintain at 2°C to 8°C until use and for a maximum

of 8 days after sampling. After use or after the 8‑day period, freeze below −16°C for storage up to 1 year

or below −70°C for storage longer than 1 year.

Note: In France, store blood samples according to Appendix A, “Blood storage guidelines applicable to

France”.

Serum

Collect sera in dry tubes. Following collection, maintain at 2°C to 8°C until use and for a maximum of 8

days after sampling. After use or after the 8-day period, freeze below −16°C for storage up to 1 year or

below −70°C for storage longer than 1 year.

Ear biopsies

Sample type Storage

Dry ear biopsy

After collection, maintain below −16°C until use. After use, freeze below

−16°C for storage up to 1 year or below −70°C for storage longer than

1 year.

Liquid and wet ear biopsy (in

Allﬂex™ buer)

After collection, maintain the Allﬂex™ tube containing the skin biopsy at

room temperature, protected from light, for up to 1month.

Store lysates for retesting as indicated:

•Store individual lysates at room temperature for up to 1month, or

below −16°C for long‑term storage.

•Store pooled lysates at room temperature for up to 1 week.

Chapter1Overview

Sample storage 1

VetMAX™BVDV4ALLKitSupplemental Instructions 7

Required materials not supplied

Unless otherwise indicated, all materials are available through thermoﬁsher.com. "MLS" indicates that

the material is available from ﬁsherscientiﬁc.com or another major laboratory supplier.

Catalog numbers that appear as links open the web pages for those products.

Table1Materials required for all sample preparation and extraction methods

Item Source

Equipment

Class II microbiological safety cabinet (MSC) MLS

Precision micropipettes (range of 1 µL to 1,000µL) with DNase/RNase‑free ﬁlter tips MLS

Laboratory mixer, vortex or equivalent MLS

Benchtop microcentrifuge capable of 8,000–15,000 × g (for 1.5‑mL and 2‑mL

microtubes) MLS

Reagents

RNase/DNase‑free water (for negative control samples (NCS)) MLS

Consumables

1.5‑mL DNase/RNase‑free microtubes MLS

Table2Additional materials required for automated magnetic bead extraction

Item Source

Instrument and equipment

One of the following instruments[1]:

•KingFisher™ Flex Puriﬁcation System

•MagMAX™ Express‑96 Magnetic Particle Processor

•KingFisher™ Duo Prime Puriﬁcation System

•KingFisher™ mL Food Protection Puriﬁcation System

•MagMAX™ Express-24 Magnetic Particle Processor

Contact your local sales

oce.

Kits and reagents[1]

One of the following kits:

•MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

•MagMAX™-96 Viral RNA Isolation Kit

•A32700 or A32702

•AM1836

Isopropanol (100%)[2] MLS

[1] Depending on the extraction protocol used.

[2] Required for use with the MagMAX™-96 Viral RNA Isolation Kit only.

Chapter1Overview

Required materials not supplied

8VetMAX™BVDV4ALLKitSupplemental Instructions

Table3Additional materials required for manual extraction on silica columns (blood and serum samples

only)

Item Source

Kits and reagents

One of the following kits:

•QIAamp™ Viral RNA Mini Kit

•NucleoSpin™ RNA Virus kit

•QIAGEN™

•MACHEREY‑NAGEL

Table4Additional materials required for manual extraction on silica columns (ear biopsies only)

Item Source

Kits and reagents

One of the following kits:

•RNeasy™MiniKit

•NucleoSpin™RNA kit

•QIAGEN™

•MACHEREY‑NAGEL

Consumables

QIAshredder[1] QIAGEN™

[1] Required for use with the RNeasy™MiniKit only.

Table5Additional materials required for rapid lysis of ear biopsies

Item Source

Equipment

Incubator or heat block set to 95°C[1] MLS

Two Incubators or heat blocks set to 70°C and 90°C[2] MLS

Kits and reagents

VetMAX™ Ear Notch Fast Lysis Kit FLK

Consumables

Microtubes (compatible with the incubator or heat block that is used) MLS

[1] Required for the One‑Step Heating Workflow.

[2] Required for the Two‑Step Heating Workflow.

Chapter1Overview

Required materials not supplied 1

VetMAX™BVDV4ALLKitSupplemental Instructions 9

Recommended RNA extraction protocols

Recommended extraction kits

Whole blood, serum MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

MagMAX™-96 Viral RNA Isolation Kit

QIAamp™ Viral RNA Mini Kit

NucleoSpin™ RNA Virus kit

Ear biopsies MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

MagMAX™-96 Viral RNA Isolation Kit

RNeasy™MiniKit

NucleoSpin™RNA kit

Ear biopsies (rapid lysis) VetMAX™ Ear Notch Fast Lysis Kit

Chapter1Overview

Recommended RNA extraction protocols

10 VetMAX™BVDV4ALLKitSupplemental Instructions

RNA extraction from EDTA blood,

serum

■Preparation of samples before extraction ................................................ 11

■Guidelines for exogenous Internal Positive Control (IPC)use ............................... 12

■Extraction with the MagMAX™ CORE Nucleic Acid PuriﬁcationKit .......................... 12

■Extraction with the MagMAX™‑96 Viral RNA IsolationKit .................................. 20

■Extraction with the QIAamp™ Viral RNA MiniKit .......................................... 22

■Extraction with the NucleoSpin™ RNA Viruskit ........................................... 23

Preparation of samples before extraction

Whole blood collected in EDTA tubes

Whole blood collected in EDTA tubes is used directly, without preparation prior to extraction.

The extraction requires 50–200µL of blood.

Serum for individual analysis

Serum, issued from blood collected in dry tubes, is used directly, without preparation prior to

extraction.

The extraction requires 50–200µL of serum.

Pooled serum or whole blood analyses

The pools may be made from dierent matrices. However, never mix blood collected in heparin

tubes with another matrix.

Pool samples manually or by machine. To limit the number of retests in the case of positive pools, ﬁrst

combine individual samples into sub-pools, then combine sub-pools into a larger pool.

To avoid contamination in the case of manual pooling, the pipette tip must be changed after the

addition of each individual sample and each constituent of the sample pool.

In the case of automatic pooling, the risk of contamination between wells by the machine must

be checked according to the manufacturer’s recommendations.

VetMAX™BVDV4ALLKitSupplemental Instructions 11

For each analyzed pool:

•If the result of the analysis is negative: all samples in the pool will be considered negative.

•If the result of the analysis is positive: test all sub-pools included in the pool or each sample

individually if no sub-pools were made.

Similarly, for each subsequent sub-pool:

•If the result of the analysis is negative: all samples in the sub-pool will be considered negative.

•If the result of the analysis is positive: test all samples included in the sub-pool individually.

Guidelines for exogenous Internal Positive Control (IPC) use

Serum samples require an exogenous IPC source (5 - IPC BVDV 4ALL) that is added to the lysis buer

during RNA extraction. The IPC target is already present in the blood samples, so addition of 5 - IPC

BVDV 4ALL is not required for these samples.

•For extraction series that include only serum samples, add 5 µL of 5 - IPC BVDV 4ALL to the lysis

buer during RNA extraction of the samples, BVDV positive control, and the negative extraction

control sample (NCS).

•For extraction series that include only blood samples, prepare lysis solution without 5 - IPC BVDV

4ALL for the blood samples, the BVDV positive control, and the NCS.

•For extraction series that include both serum and blood samples:

–Add 5 µL of 5 - IPC BVDV 4ALL to the lysis solution for all the serum samples, the BVDV

positive control, and the NCS.

–Prepare lysis solution without 5 - IPC BVDV 4ALL for the blood samples in the extraction

series.

Extraction with the MagMAX™ CORE Nucleic Acid

Puriﬁcation Kit

Recommended workﬂows

Two workﬂow options are available for the extraction of blood and serum samples using the MagMAX™

CORE Nucleic Acid Puriﬁcation Kit:

•Multisample Simple Workﬂow—Recommended for processing a combination of sample types.

•Simple Workﬂow—Recommended for processing blood and serum samples only.

Note: The Multisample Simple and Simple Workﬂows dier only in the order that the reagents are

added to the samples.

Chapter2RNA extraction from EDTA blood, serum

Guidelines for exogenous Internal Positive Control (IPC) use

12 VetMAX™BVDV4ALLKitSupplemental Instructions

Workﬂow: Multisample Simple

Start with serum or whole blood samples (individual or pooled)

▼

Follow the appropriate procedure based on your instrument:

KingFisher™ Flex or MagMAX™ Express-96 KingFisher™ Duo Prime or KingFisher™mL

Set up the processing plates (page15)

▼

Prepare samples for processing using the

Multisample Simple Workﬂow (page16)

Prepare Lysis/Binding/Bead Mix (page16)

Prepare the sample (page16)

Combine the sample with PK, then add Lysis/

Binding/Bead Mix (page16)

▼

Process samples on the instrument (page19)

Prepare samples for processing using the

Multisample Simple Workﬂow (page16)

Prepare Lysis/Binding/Bead Mix (page16)

Prepare the sample (page16)

Combine the sample with PK, then add Lysis/

Binding/Bead Mix (page16)

▼

Set up the processing plates (page19)

▼

Process samples on the instrument (page19)

Workﬂow: Simple

Start with serum or whole blood samples (individual or pooled)

▼

Follow the appropriate procedure based on your instrument:

KingFisher™ Flex or MagMAX™ Express-96 KingFisher™ Duo Prime or KingFisher™mL

Set up the processing plates (page15)

▼

Prepare samples for processing using the

Simple Workﬂow (page17)

Prepare Bead/PKMix (page17)

Prepare Lysis/Binding Solution (page17)

Prepare the sample (page18)

Combine the sample with Bead/PKMix, then add

Lysis/Binding Solution (page18)

▼

Process samples on the instrument (page19)

Prepare samples for processing using the Simple

Workﬂow (page17)

Prepare Bead/PKMix (page17)

Prepare Lysis/Binding Solution (page17)

Prepare the sample (page18)

Combine the sample with Bead/PKMix, then add

Lysis/Binding Solution (page18)

▼

Set up the processing plates (page19)

▼

Process samples on the instrument (page19)

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit 2

VetMAX™BVDV4ALLKitSupplemental Instructions 13

Procedural guidelines

•Before use, invert bottles of solutions and buers to ensure thorough mixing.

•Mix samples with reagents using a plate shaker or by pipetting up and down.

Note: Do not use a plate shaker with the tube strips required by the KingFisher™mL instrument.

•To prevent cross-contamination:

–Cover the plate or tube strip during the incubation and shaking steps, to prevent spill-over.

–Carefully pipet reagents and samples, to avoid splashing.

•To prevent nuclease contamination:

–Wear laboratory gloves during the procedures. Gloves protect you from the reagents, and they

protect the nucleic acid from nucleases that are present on skin.

–Use nucleic acid-free pipette tips to handle the reagents, and avoid putting used tips into the

reagent containers.

–Decontaminate lab benches and pipettes before you begin.

Before ﬁrst use of the kit

(Optional) Determine the maximum plate shaker setting

If a plate shaker is used, determine the maximum setting:

1. Verify that the plate ﬁts securely on your shaker.

2. Add 1 mL of water to each well of the plate, then cover with sealing foil.

3. Determine the maximum setting that you can use on your shaker without any of the water

splashing onto the sealing foil.

Download the script

The scripts for the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit are not pre-installed on the

instruments.

1. On the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit product web page (at thermoﬁsher.com,

search by catalogue number), scroll to the Product Literature section.

2. Right‑click the appropriate ﬁle to download the latest version of the MagMAX_CORE script for your

instrument.

Table6Recommended scripts

Instrument Script name

KingFisher™ Flex MagMAX_CORE_Flex.bdz

KingFisher™96

MagMAX™ Express-96

MagMAX_CORE_KF-96.bdz

KingFisher™ Duo Prime MagMAX_CORE_DUO.bdz

KingFisher™mL MagMAX_CORE_mL_no_heat.bdz

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

14 VetMAX™BVDV4ALLKitSupplemental Instructions

If required by your laboratory, use one of the following scripts, which do not heat the samples

during the elution step.

Table7Alternate scripts without heated elution step

Instrument Script name

KingFisher™ Flex MagMAX_CORE_Flex_no_heat.bdz

KingFisher™96

MagMAX™ Express-96

MagMAX_CORE_KF-96_no_heat.bdz

KingFisher™ Duo Prime MagMAX_CORE_DUO_no_heat.bdz

KingFisher™mL MagMAX_CORE_mL_no_heat.bdz

See your instrument user guide or contact Technical Support for instructions for installing the script.

Set up the processing plates (KingFisher™ Flex and MagMAX™ Express-96

instruments)

IMPORTANT! If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, do not set up

the processing plates or tube strips before preparing the samples.

1. Set up the processing plates.

Table8Plate setup: KingFisher™ Flex or MagMAX™ Express-96 instrument

Plate ID Plate

position[1] Plate type Reagent Volume per

well

Wash Plate 1 2 Deep Well MagMAX™ CORE Wash Solution 1 500µL

Wash Plate 2 3 Deep Well MagMAX™ CORE Wash Solution 2 500µL

Elution 4 Standard MagMAX™ CORE Elution Buer 90µL

Tip Comb 5 Standard Place a tip comb in the plate.

[1] Position on the instrument.

2. (Optional) To prevent evaporation and contamination, cover the prepared processing plates with

sealing foil until they are loaded into the instrument.

3. Proceed to the workﬂow that is appropriate for your laboratory.

•Multisample Simple Workﬂow—See “Prepare samples for processing using the Multisample

Simple Workﬂow” on page16.

•Simple Workﬂow—See “Prepare samples for processing using the Simple Workﬂow” on

page17.

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit 2

VetMAX™BVDV4ALLKitSupplemental Instructions 15

Prepare samples for processing using the Multisample Simple Workﬂow

Prepare Lysis/Binding/Bead Mix

1. Combine the following components, in the order indicated, for the required number of samples

plus 10% overage.

Component Volume per sample

MagMAX™ CORE Lysis Solution 350µL

MagMAX™ CORE Binding Solution 350 µL

MagMAX™ CORE Magnetic Beads 20µL

Total Lysis/Binding/Bead Mix (−IPC) 720µL

5-IPC BVDV 4ALL[1] 5 µL

Total Lysis/Binding/Bead Mix (+IPC) 725µL

[1] Add 5-IPC BVDV 4ALL only to serum samples, and to BVDV positive control and NCS in extraction series that include at least

one serum sample.

2. Mix by inverting the tube or bottle at least 10times.

Prepare the sample

Prepare samples and controls as described.

Sample type Action

Individual samples Proceed with 200µL of sample.

Pooled samples Proceed with 200µL of pooled samples.[1]

BVDV positive control Proceed with 50µL of 4c-EPC BVDV 4ALL.

NCS —

[1] For information on the preparation of samples before extraction, see “Pooled serum or whole blood analyses” on page11.

Combine the sample with PK, then add Lysis/Binding/Bead Mix

1. Add 10µL of MagMAX™ CORE Proteinase K to the required wells in the plate or tube strip.

2. Transfer each prepared sample or control to a well with MagMAX™ CORE Proteinase K.

3. Mix the sample with Proteinase K for 2 minutes at room temperature according to your mixing

method.

•Using a plate shaker: shake vigorously for 2 minutes (see “(Optional) Determine the maximum

plate shaker setting” on page14).

•By pipetting: pipet up and down 3 times, then incubate for 2 minutes at room temperature.

(For downstream processing on the KingFisher™mL instrument, you must mix by pipetting.)

4. Invert the tube of Lysis/Binding/Bead Mix several times to resuspend the beads, then add 720µL

of Lysis/Binding/Bead Mix to each sample.

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

16 VetMAX™BVDV4ALLKitSupplemental Instructions

5. Immediately proceed to “Process samples on the instrument” on page19.

Note: If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, proceed to “Set

up the processing plates or tube strips (KingFisher™ Duo Prime and KingFisher™mL instruments)”

on page19.

Prepare samples for processing using the Simple Workﬂow

Prepare Bead/PKMix

We recommend that you prepare new Bead/PK Mix for each processing run. If necessary, you can store

Bead/PK Mix at 4°C for up to 1 week.

1. Vortex the MagMAX™ CORE Magnetic Beads thoroughly to ensure that the beads are fully

resuspended.

2. Combine the following components for the required number of samples, plus 10% overage

(recommended).

Component Volume per sample

MagMAX™ CORE Magnetic Beads 20µL

MagMAX™ CORE Proteinase K 10µL

Total Bead/PKMix 30µL

Prepare Lysis/Binding Solution

1. Combine the following components for the required number of samples plus 10% overage.

Component Volume per sample

MagMAX™ CORE Lysis Solution 350µL

MagMAX™ CORE Binding Solution 350 µL

Total Lysis/Binding Solution (–IPC) 700µL

5-IPC BVDV 4ALL[1] 5 µL

Total Lysis/Binding Solution (+IPC) 705µL

[1] Add 5-IPC BVDV 4ALL only to serum samples, and to BVDV positive control and NCS in extraction series that include at least

one serum sample.

2. Mix by inverting the tube or bottle at least 10times.

(Optional) Store Lysis/Binding Solution at room temperature for up to 24 hours.

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit 2

VetMAX™BVDV4ALLKitSupplemental Instructions 17

Prepare the sample

Prepare samples and controls as described.

Sample type Action

Individual samples Proceed with 200µL of sample.

Pooled samples Proceed with 200µL of pooled samples.[1]

BVDV positive control Proceed with 50µL of 4c-EPC BVDV 4ALL.

NCS —

[1] For information on the preparation of samples before extraction, see “Pooled serum or whole blood analyses” on page11.

Combine the sample with Bead/PK Mix, then add Lysis/Binding Solution

1. Invert the tube of Bead/PK Mix several times to resuspend the beads, then add 30µL of the

Bead/PK Mix to the required wells in the plate or tube strip.

2. Transfer each prepared sample to a well with Bead/PKMix.

3. Mix the sample with Bead/PK Mix for 2 minutes at room temperature according to your mixing

method.

•Using a plate shaker: shake vigorously for 2 minutes (see “(Optional) Determine the maximum

plate shaker setting” on page14).

•By pipetting: pipet up and down 3 times, then incubate for 2 minutes at room temperature.

(For downstream processing on the KingFisher™mL instrument, you must mix by pipetting.)

4. Add 700 µL of Lysis/Binding Solution to each sample-containing well or tube strip.

5. Immediately proceed to “Process samples on the instrument” on page19.

Note: If you are using the KingFisher™ Duo Prime or KingFisher™mL instrument, proceed to “Set

up the processing plates or tube strips (KingFisher™ Duo Prime and KingFisher™mL instruments)”

on page19.

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit

18 VetMAX™BVDV4ALLKitSupplemental Instructions

Set up the processing plates or tube strips (KingFisher™ Duo Prime and

KingFisher™mL instruments)

Note: When performing this procedure for processing on the KingFisher™mL instrument, mix samples

by pipetting up and down. Do not use a plate shaker with the tube strips required by this instrument.

1. Add each reagent to the indicated positions, according to your instrument.

Load the Tip Comb and all of the plates or tube strips at the same time. The instrument does not

prompt you to load items individually.

Table9Plate setup: KingFisher™ Duo Prime instrument

Row ID Row in the plate Plate type Reagent Volume per well

Sample A Deep Well Sample lysate/bead mix Varies by sample

Wash 1 B MagMAX™ CORE Wash Solution

500µL

Wash 2 C MagMAX™ CORE Wash Solution

500µL

Elution[1] Separate tube

strip[2]

Elution strip MagMAX™ CORE Elution Buer 90µL

Tip Comb H Deep Well Place a tip comb in the plate.

[1] Ensure that the elution strip is placed in the correct direction in the elution block.

[2] Placed on the heating element.

Table10Tube strip setup: KingFisher™mL instrument

Position ID Tube strip

position Tube Reagent Volume per well

Sample 1 Standard Sample lysate/bead mix Varies by sample

Wash 1 2 MagMAX™ CORE Wash Solution

500µL

Wash 2 3 MagMAX™ CORE Wash Solution

500µL

Elution 4 MagMAX™ CORE Elution Buer 90µL

Tip Comb N/A N/A Slide the tip comb into the tip comb holder.

2. Immediately proceed to “Process samples on the instrument” on page19.

Process samples on the instrument

1. Select the appropriate script on the instrument (see “Download the script” on page14).

2. Start the run, then load the prepared plates or tube strips in their positions when prompted by the

instrument.

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™ CORE Nucleic Acid Puriﬁcation Kit 2

VetMAX™BVDV4ALLKitSupplemental Instructions 19

Store puriﬁed nucleic acid on ice for immediate use, at −20°C for up to 1month, or at −80°C for

long‑term storage.

Extraction with the MagMAX™‑96 Viral RNA Isolation Kit

Notes

The extraction protocol on magnetic beads given here is used with the MagMAX™ Express-24 Magnetic

Particle Processor.

Before starting

•Preparation of the TL lysis buer. Reconstitute the lysis buer as described below:

Components For 1 sample For N samples[1]

Lysis/Binding Solution

Concentrate

70µL N×70µL

Carrier RNA[2] 1µL N×1 µL

Vortex brieﬂy

Add 100% isopropanol 70µL N×70µL

Vortex

[1] It is recommended to allow for an additional reaction with respect to the total number of extractions to be carried out (samples

+ controls).

[2] The carrier RNA may have a viscous appearance once thawed, which makes pipetting more difficult. In this case, place the

tube at 37°C for 10 to 15 minutes, vortex vigorously and centrifuge.

Once reconstituted, the TL lysis buer is stable for 1 month at room temperature; do not store

it at 2°C to 8°C as the carrier RNA may precipitate. If the TL is kept at 2°C to 8°C by mistake,

incubate for 10 to 15 minutes at 37°C and completely homogenize before use.

•For extraction series that include only serum samples, or blood samples and serum samples:

prepare TL+IPC lysis solution by adding 5 µL of 5 - IPC BVDV 4ALL to 140 µL of previously

prepared TL lysis buer. Prepare this solution just before use including an additional 10%. Discard

after use.

•Reconstitution of Wash buer 1: add the required quantity of 100% isopropanol to the Wash

Solution 1 Concentrate bottle according to the manufacturer’s recommendations prior to initial use.

•Reconstitution of Wash buer 2: add the required quantity of 96–100% ethanol to the Wash

Solution 2 Concentrate bottle according to the manufacturer’s recommendations prior to initial use.

•Reconstitution of the Mix Beads solution: each reaction requires 10µL of RNA Binding Beads and

10µL of Lysis/Binding Enhancer. This solution should be reconstituted just before use and then

discarded. Completely homogenize by gentle agitation and place at 2°C to 8°C until use.

•Prepare and identify the number of microtubes and extraction plates according to the number of

samples to be extracted (including negative and positive controls).

Chapter2RNA extraction from EDTA blood, serum

Extraction with the MagMAX™‑96 Viral RNA Isolation Kit

20 VetMAX™BVDV4ALLKitSupplemental Instructions

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Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

Brand: Thermo Fisher Scientific

Model: VetMAX BVDV 4ALL Kit

Type: Operating instructions

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Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

Thermo Fisher Scientific VetMAX BVDV 4ALL Kit Operating instructions

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