Instructions for Use - GBO · fr Ne pas réutiliser Date limite de conser-vation jusqu’au Lire...

oCheck® DNA Extraction Kit

Instructions for Use

Single Column Preparation (Cat. No. 515 040)8 Columns Preparation (Cat. No. 515 050)

For in-vitro diagnostic use by professional laboratory personnel only Revision 01 / November 2011

Greiner Bio-One GmbH Maybachstr. 2 • 72636 Frickenhausen • Germany Phone: +49 (0) 7022 948-0 • Fax +49 (0) 7022 948-514 [email protected] • www.gbo.com/bioscience

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oCheck® DNA Extraction Kit - Instructions for UseRevision 01 / November 2011

2

GLOSSARY OF SYMBOLS

en Do not reuse

Use by Consult Instruc-tions for Use

Catalog Number

Manufac-turer

In Vitro Diag-nostic Me-dical Device

Tempe-rature limitation

Contents sufficient for <n> tests

Irritant Batch code

Important Note

de Nicht wieder-verwen-den

Mindes-tens haltbar bis

Vor Gebrauch Anwei-sung lesen

Katalog-nummer

Hersteller In-Vitro- Diagnos-tika Medi-zinprodukt

Tempera-turbegren-zung

Inhalt ausrei-chend für <n> Tests

Reizend Chargen-bezeich-nung

Wichtiger Hinweis

fr Ne pas réutiliser

Date limite de conser-vation jusqu’au

Lire les in-structions avant utilisation

Numéro de réfé-rence

Fabricant Produit médi-cal de diagnostic in-vitro

Limite de tempéra-ture

Contenu suffisant pour <n> tests

irritant No de lot Note im-portante

es No utilizar más

A utilizar preferib-lemente antes de

Antes de usar, lea las instruccio-nes

Número de catálo-go

Fabri-cante

Producto medicinal de diagnósti-ca in vitro

Limitación de tempera-tura

Contenido suficiente para <n> ensayos

irritante Código de lote

Nota im-portante

it Non riutilizza-bile

Da utilizzare entro e non oltre

Leggere le istruzi-oni prima dell’uso

Numero catalogo

Produt-tore

Dispositivo medico- diagnosti-co in-vitro

Limitazio-ne tempe-ratura

Contenuto sufficiente per test <n>

Irritante Codice del lotto

Nota im-portante

pt Não utilz-ar mais

A utilizar preferí-velmente antes de

Antes de usar, leia as inst-ruções

Número de catálo-go

Fabri-cante

Producto medicinal de diagnósti-ca in vitro

Limitação de tempera-tura

Conteúdo suficiente para <n> ensaios

irritante Código do lote

Aviso im-portante

nl Niet opnieuw gebruiken

Tenminste houdbaar tot

Gebruik-saanwij-zing lezen

Catalo-gusnum-mer

Fabrikant In vitro diag-nostisch medisch product

Tempera-tuurbeper-king

Voldoen-de inhoud voor <n> tests

Irriterend Lot nummer

Belangri-jke op-merking

da Må ikke genbru-ges

Anvendes senest

Læs brugsan-visningen

Katalog-nummer

Producent In vitro meskdicin doag-nose-apparat

Tempera-turbegra-ensær

Indehol-der nok til <n> test

Lokalirri-terende

Lotnum-mer

Vigtig henvis-ning

sv Får ej återan-vändas

Sista för-bruknings-dag

Läs bruk-sanvisnin-gen före använd-ning

Katalog-nummer

Tillverkare In vitro- medicinsk doag-nostisk apparatur

Tempe-ratur-be-gränsning

Innehållet räcker till <n> tester

irriterande Lot nummer

Viktigt medde-lande

pl Nie stosować ponownie

Termin zydatności

Przed użyciem przeczytać instrukcję

Numer katalo-gowy

Producent Diag-nostykain vitroProdukt yw

Ograni-czenie tempera-tury

Zawartość wystarcza na <n> testów

drażniący Kod partii Ważne

no Må ikke brukes flere ganger

holdbar til Les bruksan-visning før bruk

katalog-nummer

produsent in vitro-di-agnostisk medisinsk utstyr

tempe-raturbe-grensning

Innhold tilstrekke-lig for <n> tester

irriterende batch nr. Viktig merknad

el Δεν ξαναχρησι μοποιείται ποιείταείται

το λιγότερο διατηρείται

πριν την χρήση διαβάστε τις οδηγίες

Αριθμός Καταλόγου

Παραγωγός In vitro διαγνωστικά ιατρικά προϊόντα

περιoριoμός θερμοκραο ίας

Περιεχόμενο αροκετό για <n> τεοτ

ερεθιστικό κωδικός παρτίδας

Σημαντική υπόδειξη

tr Yeniden ullanmayın

Son kullanma tarihi:

Kullanma-dan önce talimatı okuyun

Katalog numarası

Üretici firma

In vitro diagnostik tıbbi tanı ürünü

Sıcaklık sınırlaması

İçeriği <n> test için yeter-lidir

Tahriş edici

Parti kodu Önemli Not

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TABLE OF CONTENTS

1. KIT CONTENTS .......................................................................................................... 52. CONSUMABLES AND EQUIPMENT REQUIRED ...................................................... 6 2.1 Single Column Preparation (Cat. No. 515 040) .............................................................................6 2.2 8-Columns Preparation (Cat. No. 515 050) ....................................................................................73. SHIPMENT AND STORAGE ....................................................................................... 84. SAFETY INSTRUCTIONS ........................................................................................... 85. WASTE DISPOSAL ..................................................................................................... 96. INTRODUCTION ....................................................................................................... 10 6.1 Intended use ..................................................................................................................................10 6.2 Misuse ............................................................................................................................................10 6.3 Sample material ............................................................................................................................. 11 6.4BasicprincipleofDNApurification ............................................................................................. 117. INSTRUCTIONS FOR THE oCheck® WORKFLOW ................................................. 12 7.1 General instructions ....................................................................................................................12 7.2 Room separation ...........................................................................................................................12 7.3 Warnings and precautions ...........................................................................................................13

7.3.1 Contamination prevention ....................................................................................................13 7.3.2 General precautions ...........................................................................................................13 7.3.3 Working safely ....................................................................................................................14

8. SINGLE COLUMN PREPARATION (Cat. No. 515 040) ........................................... 15 8.1 Preparation of working solutions and storage conditions ........................................................15 8.2 Sample preparation .......................................................................................................................16

8.2.1 Sample preparation for PapilloCheck® and PapilloCheck® high-risk ................................16 8.2.2 Sample preparation for PelvoCheck® CT/NG ....................................................................16

8.3 Standard protocol .........................................................................................................................17 8.3.1 Preparation .......................................................................................................................17 8.3.2 Pre-lysis ..............................................................................................................................17 8.3.3 Lysis .................................................................................................................................17 8.3.4 Adjust DNA binding conditions ..........................................................................................17 8.3.5 Bind DNA ............................................................................................................................18 8.3.6 Wash Spin Columns ...........................................................................................................18 8.3.7 Dry Spin Columns ............................................................................................................18 8.3.8 DNA elution ........................................................................................................................19


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9. INSTALLATION AND WORKING GUIDE FOR oCheck® VAC SET AND oCheck® VAC PUMP................................................. 20 9.1 Contents .........................................................................................................................................20

9.1.1 oCheck® VacSet .................................................................................................................20 9.1.2 oCheck® VacPump .............................................................................................................21

9.2 Assembly of the oCheck® VacSet ................................................................................................22 9.2.1 Set-up for DNA binding and washing .................................................................................22 9.2.2 Set-up for DNA elution ........................................................................................................23

9.3 Assembly of the oCheck® VacPump ............................................................................................24 9.4 Setting up the vacuum ..................................................................................................................24 9.5 Apply the vacuum .........................................................................................................................24 9.6 Ventilate the manifold ...................................................................................................................2410. 8-COLUMNS PREPARATION (Cat. No. 515 050) .................................................... 25 10.1 Preparation of working solutions and storage conditions ......................................................25 10.2 Sample preparation .....................................................................................................................26

10.2.1 Sample preparation for PapilloCheck® and PapilloCheck® high-risk ................................26

10.3 Standard protocol .......................................................................................................................27 10.3.1 Preparation .........................................................................................................................27 10.3.2 Pre-lysis ..............................................................................................................................27 10.3.3 Lysis ...................................................................................................................................27 10.3.4 Adjust DNA binding conditions ...........................................................................................27 10.3.5 Bind DNA ............................................................................................................................28 10.3.6 Wash Binding Strips ...........................................................................................................30 10.3.7 Dry Binding Strips ...............................................................................................................31 10.3.8 Elution of DNA ....................................................................................................................31

11. TROUBLESHOOTING .............................................................................................. 3312. TECHNICAL ASSISTANCE....................................................................................... 3413. PERFORMANCE CHARACTERISTICS ................................................................... 3414. SHORT PROTOCOLS ............................................................................................... 35 14.1 Short protocol for Single Column Preparation (Cat. No. 515 040)..........................................35 14.2 Short protocol for 8 Columns Preparation (Cat. No. 515 050) ................................................37


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1. KIT CONTENTS

oCheck® DNA Extraction Kit Name

Single Column Preparation

(Cat. No. 515 040)

8 Columns Preparation

(Cat. No. 515 050)

Buffer L1 20 ml 20 ml

Buffer L2 12 ml 24 ml

Reagent L3 3 ml 6 ml

Buffer W1 30 ml 75 ml

Buffer W2 (concentrate) 14 ml 50 ml

Buffer E 15 ml 50 ml

Proteinase K (lyophilised) 30 mg 75 mg

Proteinase Buffer PKB 1.8 ml 3.6 ml

Carrier RNA (lyophilised) 0.3 mg 0.3 mg

Spin Columns 50 -

Collection Tubes 250 -

Binding Strips - 12

Paper Sheet - 10

Label for Buffer L4 1 1

Download Instructions for Manual 1 1

BUF L1

BUF L2

REAG L3

BUF W1

BUF W2

BUF E

BUF L4

Proteinase K

Carrier RNA

Spin Columns

Collection Tubes

Binding Strips

Paper Sheet

Proteinase BUF PKB


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2. CONSUMABLES AND EQUIPMENT REQUIRED

2.1 Single Column Preparation (Cat. No. 515 040)

Consumables Greiner Bio-One Cat. No. Quantity (pcs)

oCheck® DNA Extraction Kit – Single Column Preparation 515 040 50 preps

Sterile,DNase-freemicropipettefiltertips

0.5-10 µl filter tips 0.5-20 µl filter tips 10-100 µl filter tips10-200 µl filter tips100-1000 µl filter tips

765 288774 288772 288739 288750 288

9696969660

Additional consumables required

•1.5 or 2.0 ml reaction tubes with safe lock or screwing lock for lysis steps•Ethanol p.a. ≥ 99.8 %•Single-use gloves

Additional equipment required

•Microcentrifuge suitable for 1.5 or 2.0 ml reaction tubes and 11,000 g•Vortex mixer•Heating block, water bath or thermomixer capable for 1.5 or 2.0 ml reaction tubes for 70 °C and 56 °C incubation•Micropipettes variable from 1-1000 μl•Waste container•Timer


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2.2 8-Columns Preparation (Cat. No. 515 050)

Consumables Greiner Bio-One Cat. No. Quantity (pcs)

oCheck® DNA Extraction Kit – 8 Columns Preparation 515 050 12 x 8 preps

Sterile,DNase-freemicropipettefiltertips

0.5-10 µl filter tips 0.5-20 µl filter tips 10-100 µl filter tips10-200 µl filter tips100-1000 µl filter tips

765 288774 288772 288739 288750 288

9696969660

PCR 8-tube strips without capCap strips for 8-tube stripsSingle PCR tubes

673 210373 270671 201

125125500

Equipment Greiner Bio-One Cat. No. Quantity (pcs)

oCheck® VacSet 863 080 1

oCheck® VacPump 863 070 1

Additional consumables required

•1.5 or 2.0 ml reaction tubes with safe lock or screwing lock for lysis steps•Multi-channel pipette reservoir (disposable reagent reservoirs), volumes: 25 and 100 ml •Pipette tips (capable of 1200 μl volume) for 8-channel pipette•Ethanol p.a. ≥ 99.8 %•Single-use gloves

Additional equipment required

•Microcentrifuge suitable for 1.5 or 2.0 ml reaction tubes and 11,000 g•Vortex mixer•Heating block, water bath or thermo mixer capable for 1.5 or 2.0 ml reaction tubes for 70 °C and 56 °C incubation•Micropipettes variable from 1-1000 μl•8-channel multi pipette for large volumes (50 to 1200 μl)•Waste container•Timer•PCR rack


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3. SHIPMENT AND STORAGE

The storage and the shipment of the oCheck® DNA Extraction Kit takes place at room temperature. For storage of prepared working solutions see sections 8.1 and 10.1.

4. SAFETY INSTRUCTIONS

The following components of the oCheck® DNA Extraction Kit contain hazardous contents.

Wear gloves and goggles and follow the safety instructions given in this section.

Component HazardContents

HazardSymbol

RiskPhrases

RiskPhrases

Safety Phrases

Buffer L2 Guanidine hydrochloride

Harmful if swallowed, Irritating to eyes and

skinR 22-36/38

Buffer W1Guanidine

hydrochloride + isopropanol < 25 %

Flammable, Harmful if swallowed, Irritating to eyes and

skin

R 10-22-36/38 S7-16-25

Proteinase K Proteinase K,lyophilised

Irritating to eyes, respiratory system

and skin, May cause

sensitisation byinhalation

R 36/37/38-42 S 22-24-26-36/37

* Hazard Labelling not necessary, if quantity per bottle below 125 g or ml (according to 67/548//EEC Art. 25, 1999/45/EC Art. 12 and German GefStoffV § 20 (3) and TRGS 200 7.1). For further information see Safety Data Sheet.

Risk PhrasesR10 FlammableR22 Harmful if swallowedR 36/37/38 Irritating to eyes, respiratory system and skinR 36/38 Irritating to eyes and skinR 42 May cause sensitisation by inhalation

Safety PhrasesS7 Keep container tightly closedS16 Keep away from sources of ignition - No smokingS 22 Do not breathe dustS 24 Avoid contact with the skinS 25 Avoid contact with the eyesS 26 In case of contact with eyes rinse immediately with plenty of water and seek medical adviceS 36/37 Wear suitable protective clothing and gloves

The current version of the Safety Data Sheet for this product can be downloaded from the Greiner Bio-One website: www.gbo.com/bioscience/biochips_download

*

*

*


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5. WASTE DISPOSAL

The Guanidine hydrochloride in Buffers L2 and W1 can form highly reactive compounds when combined with bleach. Bleach or acidic solutions should not be added directly to the sample-preparation waste.

Contamination of the liquid waste with residual infectious material is highly unlikely, but cannot be completely excluded. Thus, liquid waste must be considered infectious and be handled and discarded according to local safety regulations. Please carefully follow federal, state and local guidelines for waste disposal.


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6. INTRODUCTION

6.1 Intended use

The oCheck® DNA Extraction Kit (Greiner Bio-One; Cat. No. 515 040 or Cat. No. 515 050) is an in vitro diagnostic kit and is intended to be used for the preparation of DNA from samples of human origin, to be analysed with assays from the oCheck® product line.

The nucleic acid isolation methodology used by the oCheck® DNA Extraction Kit variants produces DNA suitable for direct downstream analysis by the following kits:

oCheck® DNA Extraction Kit - Single Column Preparation (Cat. No. 515 040)• PapilloCheck® (Greiner Bio-One; Cat. No. 465 060)• PapilloCheck® high-risk (Greiner Bio-One; Cat. No. 505 060)• PelvoCheck® CT/NG (Greiner Bio-One; Cat. No. 504 002)

oCheck® DNA Extraction Kit - 8 Columns Preparation (Cat. No. 515 050)• PapilloCheck® (Greiner Bio-One; Cat. No. 465 060)• PapilloCheck® high-risk (Greiner Bio-One; Cat. No. 505 060)

The use of the oCheck® DNA Extraction Kit - 8 Columns Preparation to isolate DNA for downstream analysis with PelvoCheck® CT/NG (Greiner Bio-One; Cat. No. 504 002) assay is not recommended until the validation is completed.

The oCheck® DNA Extraction Kit is intended to be used only in combination with assays from the oCheck® product line (Greiner Bio-One, Frickenhausen, Germany), which specify the use of the oCheck® DNA Extraction Kit in their instructions for use.

The oCheck® DNA Extraction Kit fulfills the requirements of the In Vitro Diagnostic Medical Device Directive (98/79/EC) and therefore displays the CE conformance mark.The product is intended for use by professional users ONLY such as technicians and physicians trained in molecular biological techniques.

6.2 Misuse

The oCheck® DNA Extraction Kit is NOT intended to be used with either CarnoCheck® (Greiner Bio-One), ParoCheck® (Greiner-Bio-One) or any other product not found in the oCheck® product line.


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6.3 Sample material

For the downstream application of PapilloCheck® (Greiner Bio-One; Cat. No. 465 060) and PapilloCheck® high-risk (Greiner Bio-One; Cat. No. 505 060) the oCheck® DNA Extraction Kit was designed and tested to extract viral and human genomic DNA from human cervical smears collected with one of the following sample collection systems:

• PapilloCheck® Collection Kit (Greiner Bio-One; Cat. No. 465 070)• PreservCyt® (Hologic, Bedford, MA, USA)• SurepathTM (BD, Franklin Lakes, NJ, USA)• STMTM (Qiagen, Hilden, Germany)

For the downstream application of the PelvoCheck® CT/NG (Greiner Bio-One; Cat. No. 504 002) assay the oCheck® DNA Extraction Kit was validated for the extraction of bacterial and human genomic DNA from human urine samples using the following collection systems:

• PelvoCheck® Collection Kit SAFE (Greiner Bio-One; Cat. No. 453 100)• PelvoCheck® Collection Kit STRAW (Greiner Bio-One; Cat. No. 453 101)

Other sample collection systems have not been validated and must not be used with the oCheck® DNA Extraction Kit.

6.4 BasicprincipleofDNApurification

The oCheck® DNA Extraction Kit is designed for the purification of viral, bacterial and human genomic DNA from samples of human origin. The sample material is quickly and efficiently lysed via the addition of Proteinase K and Buffer L1. Lysis buffer and ethanol create appropriate conditions for binding of nucleic acids to the silica membrane of the Binding Columns. Carrier RNA improves binding and recovery of low concentrated viral, bacterial and genomic DNA. Contaminants (potential PCR inhibitors) such as salts, metabolites and soluble macromolecular cellular components are removed during washing steps with ethanolic wash buffers. Pure bacterial, viral and human genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer. After purification, the DNA is in a suitable solution for direct analysis by assays from the oCheck® product line.


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7. INSTRUCTIONS FOR THE oCheck® WORKFLOW

7.1 General instructions

The following instructions are based on laboratory experience with the implementation of presently used state-of-the-art techniques in molecular biology and aim to ensure a maximum of safety for PCR-based methods. Reasonable precautions should be taken when using the oCheck® DNA Extraction Kit in combination with further downstream analysis of the purified DNA with assays from the oCheck® product line:

In general, molecular biology laboratory technique such as DNA extraction, amplification and detection requires appropriately qualified personnel. In addition, a clean and well-structured workflow is necessary to prevent erroneous results, due to degradation or contamination by amplification products of the nucleic acid sample. To minimise these risks, it is necessary to separate the areas of extraction and amplification.

Never introduce an amplification product into the area which has been designated for extraction only. Both areas should be equipped with separate equipment, consumables, lab coats and gloves. Never transfer lab coats, gloves or equipment from the area intended for extraction to the area intended for amplification.

7.2 Room separation

Figure 1 shows an example of how a laboratory may be separated into 3 distinct sections. One is designated for DNA extraction solely, another is for the set up and running of PCR reactions and the last is for hybridisation and analysis. Each room is to be used exclusively for the application or technique indicated to prevent sample contamination. The use of colour coding could be advantageous in avoiding the accidental exchange of equipment and consumables between areas.

Neither equipment nor consumables should be interchanged between the different laboratory rooms and spaces. Hence, duplications in equipment and consumables are a necessity and have to be taken into account.

Figure 1: Room Separation in the oCheck® workflowRoom 1: DNA extraction, Room 2: Set-up of PCR reaction and division of the reaction mix in a clean bench; separate space for the addition of the template DNA; Room 3: Hybridisation and analysis

Room 1 Room 2 Room 3


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The entire DNA extraction procedure of the oCheck® DNA Extraction Kit has to be performed in room 1, the DNA extraction room. After leaving this room lab coats must be changed. Within the oCheck® workflow, DNA extraction is the process most sensitive to contamination and so strict adherence to the instructions contained within this document is essential. In addition to this, contamination prevention guidelines outlined in 7.3.1 should be followed.

Within room 2, the reaction mix for the PCR is set up and aliquoted on a clean bench. The addition of the DNA extracted in room 1 must be carried out in a separate space within room 2. After leaving this room, lab coats must be changed again.

Within the third laboratory room, the hybridisation reaction and washing steps are carried out. In addition, room 3 also contains the CheckScannerTM in conjunction with the CheckReportTMSoftware for the final analysis of the oCheck® product line assays.

7.3 Warnings and precautions

7.3.1 Contamination prevention

• Lab coats must be worn throughout the procedures and different sets of lab coats are needed for each laboratory room.

• Gloves must be worn during each step of the analysis and have to be changed for each different step.

• Lab cleanness: The working place must be decontaminated before and after work with appropriate cleaning solution.

• Reaction tubes: Never touch the inside of the cap of the reaction tube. To avoid cross-contamination, open only one tube at a time. To prevent contamination use reaction tubes with safe lock or screw lock for lysis steps.

• Appropriate micropipettefiltertips must be used (sterile, free of DNase, RNase and human DNA). Pipette tips should always be changed between liquid transfers.

7.3.2 General precautions

• Upon arrival, check the oCheck® DNA Extraction Kit components for damage. If one of the components is damaged (e.g. buffer bottles), contact your local Greiner Bio-One distributor. Do not use damaged kit components, since their use may lead to poor kit performance.

• Do not use expired reagents. • Do not use the oCheck® DNA Extraction Kit after the expiry date.• Do not mix reagents from different batches.• Use only reagents/equipment provided with the kit and those recommended by the manufacturer.• Regular calibration/check-up is necessary for pipettes, water bath and heating block.• Pipetting of small amounts of liquid in the microliter range is a challenge. Therefore, take care to

pipette with the micropipettes as accurately as possible.• All centrifuge steps should be carried out at room temperature (18-25 °C).• Unused reagents and waste material must be disposed of in accordance with federal and state

guidelines.


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7.3.3 Working safely

• This kit is for in vitro diagnostic use only and should be exclusively used by personnel trained in in vitro diagnostic laboratory practice.

• Never pipette solutions by mouth.• Do not eat, drink, smoke or apply cosmetic products in the work areas.• Take care whilst handling biological samples containing potential human infectious material. To

minimise the risk of infection from potentially infectious material, it is recommended to work under laminar air-flow conditions until the samples are lysed. Handle and dispose of all biological samples as if they were capable of transmitting infectious agents. Avoid direct contact with the biological samples as well as splashing or spraying.

• Wash hands carefully after handling samples and reagents.• Always wear lab coat, gloves and goggles while working with human samples. The current

version of the Material Safety Data Sheet for this product can be downloaded from the Greiner Bio-One website: www.gbo.com/bioscience/biochips_download


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8. SINGLE COLUMN PREPARATION (Cat. No. 515 040)

8.1 Preparation of working solutions and storage conditions

All kit components should be stored at room temperature (18-25 °C) and are stable for two years upon date of manufacture.

Before beginning with the oCheck® DNA Extraction Kit protocol, prepare the following solutions:

1. Proteinase K solution: Before first use of the kit, add 1.35 ml of Proteinase Buffer (PKB) to dissolve the lyophilised Proteinase K. Divide the solution into aliquots and store at -20 °C for up to 6 months. Do not refreeze.

2. Buffer W2: Add 56 ml of ethanol (p.a. ≥ 99,8%) to the Buffer W2 concentrate. Mark the current date on the bottle label. Buffer W2 is stable at room temperature (18-25 °C) for up to one year.

3. Buffer L4: Transfer Buffer L2 to Reagent L3 completely and mix well. Label the bottle with label L4 supplied in the kit. Mark the current date on the bottle label. The resulting Buffer L4 is stable for up to one year at room temperature (18-25 °C).

4. Carrier RNA solution: Add 900μlofBufferL1 to dissolve the lyophilised Carrier RNA. Dispense the dissolved Carrier RNA in aliquots, e.g. aliquots of 35μl for 12 extractions. Store the dissolved Carrier RNA solution at -20 °C. Do not refreeze! Used vials must be discarded once thawed.

Upon storage, especially at low temperatures, a white precipitate may form in Buffers L1, L2, or L4. Such precipitates can be easily dissolved by incubating the bottle at 50-70 °C before use.


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8.2 Sample preparation

8.2.1 Sample preparation for PapilloCheck® and PapilloCheck® high-risk

Human cervical smear samples which are collected in• PapilloCheck® Collection Kit (Greiner Bio-One; Cat.-No. 465 070)• PreservCyt® collection medium (Hologic, Bedford, MA USA)can be processed directly.

Cervical smear samples which are collected in• SurepathTM collection medium (BD, Franklin Lakes, NJ USA) must be washed before use:

centrifuge 250μl of the sample for 5 minutes at 11,000 g and resuspend the pellet in 250μl of distilled water.

Cervical smear samples which are collected in• STMTM collection medium (Qiagen, Hilden, Germany) must be diluted with distilled water: 100μl

of sample + 150μl of distilled water

In general, if the cervical smear sample is very concentrated and already aggregated, the sample must be diluted and homogenised before beginning the extraction!

Cervical smear samples which are very dilute and show no visible cells should be concentrated to achieve a higher cell extraction yield. Centrifuge up to 1000μl of sample for 5 minutes at 11,000 g and resuspend the pellet in 250μl of distilled water.

This concentration step can only be applied for cervical smear samples which were collected using either the PapilloCheck® Collection Kit, the PreservCyt® collection medium or the SurepathTM collection medium. If the STMTM collection medium was used, a concentration by centrifugation is not possible.

➠ Pipet 250μl of the sample solution into a 1.5 ml or 2 ml reaction tube. To prevent contamination use reaction tubes with safe lock or screw lock lids for all lysis steps. For purpose of validating the entire DNA isolation procedure, for each DNA extraction cycle use of a negative sample control is recommended.

8.2.2 Sample preparation for PelvoCheck® CT/NG

Human urine samples, which are collected in:• PelvoCheck® Collection Kit SAFE (Greiner Bio-One; Cat. No. 453 100)• PelvoCheck® Collection Kit STRAW (Greiner Bio-One; Cat. No. 453 101)can be directly processed.

➠ Pipet 250μl of the urine sample into a 1.5 ml or 2 ml reaction tube. Use of reaction tubes with safe lock or screw lock lids for all lysis steps is recommended. To validate the DNA extraction, a negative sample control should be used for each extraction cycle.

17oCheck® DNA Extraction Kit - Instructions for UseRevision 01 / November 2011

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8.3 Standard protocol

8.3.1 Preparation

➠ Check that the centrifuge is set at room temperature (18-25 °C). ➠ Prepare samples according to chapter 8.2. ➠ Set heat blocks or water baths to 56 °C and 70 °C, respectively. ➠ Before elution, incubate elution Buffer E at 70 °C. ➠ Make sure that working solutions have been prepared according to chapter 8.1.

8.3.2 Pre-lysis

➠ Add 80μl of Buffer L1. ➠ Add 2.4μl of prepared Carrier RNA solution. ➠ Add 20μl of Proteinase K solution. ➠ Close the lid and mix shortly by pulse-vortexing. ➠ Incubate at 56 ºC for at least 30 minutes. Shake the samples using a thermo mixer at 650 rpm.

If using a heating block or water bath, vortex the samples occasionally throughout the incubation period.

If processing several samples, Proteinase K, Buffer L1 and Carrier RNA should be premixed directly before use. Never mix Buffer L1 and Proteinase K more than 10–15 minutes before addition to the sample. Proteinase K tends to self-digestion in Buffer L1 in the absence of substrate.

8.3.3 Lysis

➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid. ➠ Add 250μl of Buffer L4. ➠ Close the lid and mix by pulse-vortexing for 10 seconds. ➠ Incubate the reaction tubes at 70 ºC for 15 minutes.

Close lids of the reaction tubes tightly to prevent accidental opening of the lids.

8.3.4 Adjust DNA binding conditions

➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid. ➠ Add 300μl of ethanol (p.a. ≥ 99,8 %) to the sample. ➠ Immediately, close the lid and mix thoroughly by pulse-vortexing for 15 seconds. ➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid.


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8.3.5 Bind DNA

To prevent contamination: put on new gloves before this step!

➠ For each sample, place one Spin Column into a Collection Tube. ➠ Carefully pipet 450 μl of the lysates into the Spin Column and centrifuge at 11,000 g for

1 minute at room temperature. Do not discard the rest of the lysate! ➠ Discard the Collection Tube containing the flow-through.


➠ Prepare new Collection Tubes and put the Spin Columns into fresh Collection Tubes. ➠ Pipette the rest of the 450μl lysate into the Spin Column. ➠ Centrifuge at 11,000 g for 1 minute at room temperature.


➠ Discard the Collection Tube with the flow-through and put the Spin Columns into fresh Collection Tubes.

8.3.6 Wash Spin Columns

➠ Apply 500μl of Buffer W1 into the Spin Column. ➠ Centrifuge at 11,000 g for 1 minute at room temperature. ➠ Discard the Collection Tube with the flow-through and place the Spin Column back into a fresh

Collection Tube. ➠ Apply 600μl of Buffer W2 into the Spin Column. ➠ Centrifuge at 11,000 g for 1 minute at room temperature. ➠ Discard the Collection Tube with the flow-through and place the Spin Column into a fresh

Collection Tube.

8.3.7 Dry Spin Columns

➠ Centrifuge at 11,000 g for 1 minute to dry the membrane completely.

This step is necessary to eliminate traces of ethanol. The ethanol in Buffer W2 inhibits enzymatic reactions and must be removed completely before DNA elution.


!

!

8.3.8 DNA elution

Use preheated (70 °C) Buffer E!

To avoid a loss of temperature during pipetting steps, we recommend filling 1.5 or 2.0 ml reaction tubes with a maximum of 1,300μlofBufferE(sufficient for n = 12 samples).


➠ Place the Spin Column into a clean 1.5 ml reaction tube. ➠ Apply 100μl of preheated (70 °C) Buffer E to the center of the membrane in the Spin Column. ➠ Close the lid and incubate at room temperature for 1 minute. ➠ Centrifuge at 11,000 g for 1 minute.

For optimal performance of isolated DNA in downstream applications, we recommend elution with the supplied Buffer E. Store the extracted DNA at -20 °C.For further analysis with assays from the oCheck® product line refer to the respective Instructions for Use (PapilloCheck®, PapilloCheck® high-risk and PelvoCheck® CT/NG).


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9. INSTALLATION AND WORKING GUIDE FOR oCheck® VAC SET AND oCheck® VAC PUMP

The oCheck® Vacuum Manifold is designed for the rapid manual parallel purification of nucleic acids using the oCheck® DNA Extraction Kit - 8 Columns Preparation. This system allows rapid simultaneous purification of high-quality nucleic acids. Time-consuming centrifugation and decanting steps are eliminated through vacuum use. The oCheck® DNA Extraction Kits are based on ultra-filtration technology and offer a fast and convenient way of nucleic acid purification.

When using the oCheck® DNA Extraction Kit - 8 Columns Preparation under vacuum, the oCheck® VacSet (Cat. No. 863 080) and the oCheck® VacPump (Cat. No. 863 070) are recommended.

9.1 Contents

9.1.1 oCheck® VacSet

Figure 2: oCheck® VacSet contents

Column Holder Microtube Rack Dummy Strips (12x)

Manifold Lid

Manifold Base Spacers

Waste Containers

Vacuum Regulator


21

9.1.2 oCheck® VacPump

Figure 3: oCheck® VacPump contents

oCheck® VacPump

Connection Tubes

Silencer


22

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9.2 Assembly of the oCheck® VacSet

➠ Place the Manifold Base on a secure lab bench. The Vacuum Manifold has to be prepared as shown in figure4a+b.

Figure 4: a) Set-up for DNA binding and washing b) Set-up for DNA elution

9.2.1 Set-up for DNA binding and washing

➠ For DNA binding and washing procedures (protocol steps 10.3.5 - 10.3.7) set up the Vacuum Manifold as shown in figure 4a.

➠ Place one of two Waste Containers* inside the Manifold Base. ➠ Close the Manifold Base with the Manifold Lid. ➠ Place the Column Holder into the rubber seal of the Manifold Lid. ➠ Insert the required amount of Binding Strips into the Column Holder. ➠ Make sure that the Binding Strips are inserted correctly and that the Bindings Strips close

tightly to the Columns Holder (closure is confirmed when a clear “click” sound is heard). If less than 48 samples are to be processed at one time, insert only the required number of Binding Strips into the holder and fill up the remaining places in the Column Holder with the appropriate amount of Dummy Strips.

Make sure that the Binding Strips and Dummy Strips fit properly into the Column Holder before applying the vacuum.* Do not place the Waste Container into a dishwasher for cleaning!

Column Holder with Binding Strips

Manifold Lid

Microtube Rack

Microtube Rack Spacer

Round-Well Block Spacer

Manifold Base


Manifold Lid

Waste Container

Manifold Base

Manifold‘s Valve


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9.2.2 Set-up for DNA elution

For the DNA elution steps (protocol step 10.3.8), the Vacuum Manifold must be modified as shown in figure4b.

➠ Remove the Column Holder together with the Binding Strips and place it on a clean Paper Sheet (provided with the kit).

➠ Remove the Manifold Lid and remove the used Waste Container. ➠ Insert Spacers “Round-Well Block” and on top “Microtube Rack” into the Manifold’s Base

short sides (figure4b). ➠ Place the Microtube Rack onto the Spacers. ➠ Place the appropriate number of labelled PCR tubes into every second row of the Microtube

Rack, starting at row No. 2 of the Microtube Rack. ➠ Close the Vacuum Manifold and place the Binding Strips on top. Check that each Binding

Strip is located directly above a PCR tube in order to collect eluted DNA (figure4c). Check again that all Binding and Dummy Strips fit correctly into the Column Holder.

Figure 4c: Each Binding Strip column must be located directly above a PCR tube from the PCR strip.


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9.3 Assembly of the oCheck® VacPump

Place the oCheck® VacPump on a secure lab bench. The oCheck® VacPump must be assembled as shown in figure 5a. To join the oCheck® VacPump to the Vacuum Manifold, use the longer tube of the two enclosed Connection Tubes. Cut the shorter Connection Tube in two parts (approx. 5 cm and 4 cm). Use the 5 cm Connection Tube to connect the oCheck VacPump to the Vacuum Regulator. Fix the Silencer on the opposite side of the oCheck® VacPump at the outlet by using the 4 cm Connection Tube.

Figure 5: a) Assembly of the oCheck® VacPump b) Vacuum Regulator

9.4 Setting up the vacuum

Make sure that the Manifold Lid, the Column Holder and the Binding Strips are placed properly. Close the Manifold’s Valve (figure5a). Start the Vacuum Pump. Adjust the vacuum according to the kit protocol by using the Vacuum Regulator (figure5b). Open the Adjusting Screw until the required vacuum is reached.

9.5 Apply the vacuum

Load the samples into the individual Binding Strips. Open the Manifold’s Valve and if necessary press down the Binding Strips shortly until the flow starts.

9.6 Ventilate the manifold

When the samples have passed through the filter of the Bindings Strips, close the Manifold’s Valve and stop the Vacuum Pump. Wait approximately 30 seconds until the Column Holder with the Bindings Strips can easily be removed.

Fix Silencer here

Manifold’s Valve Adjusting Screw


25

10. 8-COLUMNS PREPARATION (Cat. No. 515 050)

10.1 Preparation of working solutions and storage conditions

All kit components should be stored at room temperature (18-25 °C) and are stable for two years upon date of manufacture.

Before beginning with the oCheck® DNA Extraction Kit protocol, prepare the following solutions:

1. Proteinase K solution: Before first use of the kit, add 3.35 ml of Proteinase Buffer (PKB) to dissolve the lyophilised Proteinase K. Divide the solution into aliquots and store at -20 °C for up to 6 months. Do not refreeze.

2. Buffer W2: Add 200 ml of ethanol (p.a. ≥ 99,8 %) to the Buffer W2 concentrate. Mark the current date on the bottle label. Buffer W2 is stable at room temperature (18-25 °C) for up to one year.

3. Buffer L4: Transfer Buffer L2 to Reagent L3 completely and mix well. Label the bottle with label L4 supplied in the kit. Mark the current date on the bottle label. The resulting Buffer L4 is stable for up to one year at room temperature (18-25 °C).

4. Carrier RNA solution: Add 900μl of Buffer L1 to dissolve the lyophilised Carrier RNA. Dispense the dissolved Carrier RNA in aliquots, e.g. aliquots of 50μl for 16 extractions. Store the dissolved Carrier RNA solution at -20° C. Do not refreeze! Used vials must be discarded once thawed.

Upon storage, especially at low temperatures, a white precipitate may form in Buffers L1, L2, or L4. Such precipitates can be easily dissolved by incubating the bottle at 50-70 °C before use.


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10.2 Sample preparation

10.2.1 Sample preparation for PapilloCheck® and PapilloCheck® high-risk

Human cervical smear samples which are collected in• PapilloCheck® Collection Kit (Greiner Bio-One; Cat.-No. 465 070)• PreservCyt® collection medium (Hologic, Bedford, MA USA) can be directly processed.

Cervical smear samples which are collected in• SurepathTM collection medium (BD, Franklin Lakes, NJ USA) must be washed before use:

centrifuge 250μl of the sample for 5 minutes at 11,000 g and resuspend the pellet in 250μl of distilled water.

Cervical smear samples which are collected in• STMTM collection medium (Qiagen, Hilden, Germany) must be diluted with distilled water: 100μl

of sample + 150μl of distilled water.

In general, if the cervical smear sample is very concentrated and already aggregated, the sample must be diluted and homogenised before beginning the extraction!

Cervical smear samples which are very dilute and show no visible cells should be concentrated to achieve a higher cell extraytion yield. Centrifuge up to 1000μlof sample for 5 minutes at 11,000 g and resuspend the pellet in 250μl of distilled water.

This concentration step can only be applied for cervical smear samples which were collected either using the PapilloCheck® Collection Kit, the PreservCyt® collection medium or the SurepathTM collection medium. If the STMTM collection medium was used, a concentration by centrifugation is not possible.

➠ Pipet 250 μl of the sample solution into a 1.5 ml or 2 ml reaction tube. To prevent contamination use reaction tubes with safe lock or screw lock lids for all lysis steps.

For the purpose of validating the entire DNA isolation procedure, for each DNA extraction cycle use of a negative sample control is recommended.


!

10.3 Standard protocol

When using the oCheck® DNA Extraction Kit - 8 Columns Preparation - under vacuum, an appropriate vacuum system is required. The necessary equipment as well as installation and working instructions are described in chapter 9.

10.3.1 Preparation

➠ Check that the centrifuge is set at room temperature (18-25 °C). ➠ Prepare samples according to chapter 10.2 ➠ Set heat blocks or water baths to 56 °C and 70 °C, respectively. ➠ Before elution, incubate elution Buffer E at 70 °C. ➠ Make sure that working solutions have been prepared according to chapter 10.1.

10.3.2 Pre-lysis

➠ Add 80μl of Buffer L1. ➠ Add 2.4μl of prepared Carrier RNA solution. ➠ Add 20μl of Proteinase K solution. ➠ Close the lid and mix shortly by pulse-vortexing.

If processing several samples, Proteinase K, Buffer L1 and Carrier RNA should be premixed directly before use. Do not mix Buffer L1 and Proteinase K more than 10-15 minutes before addition to the sample. Proteinase K tends to self-digestion in Buffer L1 in the absence of substrate.

➠ Incubate at 56 ºC for at least 30 minutes. Shake the samples using a thermo mixer at 650 rpm. If using a heating block or water bath, vortex the samples occasionally throughout the incubation period.

10.3.3 Lysis

➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid. ➠ Add 250μl of Buffer L4. ➠ Close the lid and mix by pulse-vortexing for 10 seconds. ➠ Incubate at 70 ºC for 15 minutes. ➠ Close the lid of the reaction tubes tightly to prevent accidental lid opening.

10.3.4 Adjust DNA binding conditions

➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid. ➠ Add 300μl of ethanol (p.a. ≥ 99.8 %) to the sample. ➠ Immediately, close the lid and mix thoroughly by pulse-vortexing for 15 seconds. ➠ Briefly centrifuge the reaction tube to remove drops from the inside of the lid.


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10.3.5 Bind DNA

➠ Prepare the oCheck® Vacuum Manifold according to figure6 (refer to chapter 9 for a more detailed description).

Figure 6: Set-up for DNA binding and washing

➠ Place one of two Waste Containers* inside the Manifold Base. ➠ Close the Manifold Base with the Manifold Lid. ➠ Place the Column Holder into the rubber seal of the Manifold Lid. ➠ Insert the required amount of Binding Strips into the Column Holder. Make sure that the

Binding Strips are inserted correctly and that the Bindings Strips close tightly to the Columns Holder (closure is confirmed when a clear “click” sound is heard). If less than 48 samples are to be processed at one time, insert only the required number of Binding Strips into the holder and fill up the remaining places in the Column Holder with the appropriate number of Dummy Strips.

Make sure that the Binding Strips and Dummy Strips fit properly into the Column Holder before applying the vacuum.* Do not place the Waste Container into a dishwasher for cleaning!


Manifold Lid

Waste Container

Manifold Base

Manifold‘s Valve


➠ Carefully pipet 900μl of the lysates into the wells of the Binding Strips and be careful not to moisten the rims of the wells (figure7).

Figure 7: Pipet the lysate in the wells of the Binding Strips

➠ Set the vacuum to -200 mbar with the vacuum regulator (figure8a). ➠ Slowly apply the vacuum by opening the Manifold’s Valve until all lysates have passed through

the columns (figure8b).

Figure 8: a) Vacuum Regulator b) Manifold’s Valve

➠ Stop the vacuum directly after all of the liquid has passed the columns (approx. 30 seconds) to avoid foam formation by closing the Manifold’s Valve.

➠ Switch off the Vacuum Pump and release the vacuum.

Manifold‘s ValveAdjusting Screw


10.3.6 Wash Binding Strips

For the following steps, usage of the 8-channel pipette and reagent reservoirs is recommended (required buffer volumes if using reagent reservoirs see table 1).If using buffer reservoirs, prepare the appropriate quantity of buffer solutions.

Number of samples to extract

8 16 24 32 40 48

Buffer W1 6.5 ml 12 ml 17.5 ml 23 ml 28.5 ml 34 ml

Buffer W2* 16 ml 32 ml 48 ml 64 ml 80 ml 96 ml

Table 1: Required amounts of buffer solutions for n = x samples

* Before first use, add 200 ml ethanol (see also chapter 10.1).

➠ Add 600μl of Buffer W1 to each well of the Binding Strips (figure9a+b).

Figure 9: a) Pipet Buffer W1 from buffer reservoir. b) Addition of Buffer W1 to the wells of the Binding Strips.

➠ Set the vacuum to -200 mbar with the Vacuum Regulator. ➠ Slowly apply the vacuum by opening the Manifold’s Valve until all lysates have passed through

the columns (figure8). ➠ Stop the vacuum directly by closing the Manifold’s Valve after all of the liquid has passed the

columns (approx. 30 seconds) to avoid foam formation. ➠ Add 900μl of Buffer W2 to each well of the Binding Strips. ➠ Set the vacuum to -200 mbar with the Vacuum Regulator. ➠ Apply the vacuum until the buffer has passed through the columns (approx. 30 seconds). ➠ Stop the vacuum directly by closing the Manifold’s Valve after all of the liquid has passed the

columns (approx. 30 seconds) to avoid foam formation. ➠ Add 900μl of Buffer W2 to each well of the Binding Strips. ➠ Set the vacuum to -200 mbar with the Vacuum Regulator. ➠ Apply vacuum until all buffer has passed through the columns (approx. 30 seconds). ➠ Stop the vacuum directly by closing the Manifold’s Valve after all of the liquid has passed the

columns (approx. 30 seconds) to avoid foam formation. ➠ Switch off the Vacuum Pump and release the vacuum.


!

10.3.7 Dry Binding Strips

➠ After the final washing step stop the vacuum and ventilate the Vacuum Manifold (section 9.6). Remove the Column Holder together with the Binding Strips. Meanwhile remove the Manifold Lid and remove the used Waste Container and substitute it with the unused (clean) one.

➠ Insert the Manifold Lid together with the Column Holder and the Binding Strips into the Manifold Base. Make sure that the Manifold is closed tightly again and that all Binding Strips or Dummy Strips fit properly.

➠ Apply maximum vacuum (at least ~400 to ~600 mbar pressure difference) for 10 minutes to dry the membrane completely.

➠ Switch off the Vacuum Pump and release the vacuum.

This step is necessary to eliminate traces of ethanol. The ethanol in Buffer W2 inhibits enzymatic reactions and must be removed completely before elution of the DNA.

10.3.8 Elution of DNA

For the next elution steps the Vacuum Manifold must be modified according to figure10.

Figure 10: Set-up for DNA elution

➠ Remove the Column Holder together with the Binding Strips and place it on a clean Paper Sheet (provided with the kit).

➠ Remove the Manifold Lid and remove the used Waste Container. ➠ Insert Spacers “Round-Well Block” and on top “Microtube Rack” into the Manifold’s Base

short sides (figure10). ➠ Place the Microtube Rack onto the Spacers. ➠ Place the appropriate number of labelled PCR tubes into every second row of the Microtube

Rack starting at row No. 2 of the Microtube Rack. ➠ Close the Vacuum Manifold and place the Binding Strips on top. Check that each Binding

Strip is located directly above a PCR tube to collect the eluted DNA (figure11). Check again that all Binding and Dummy Strips still fit correctly into the Column Holder.


Manifold Lid

Microtube Rack

Microtube Rack Spacer

Round-Well Block Spacer

Manifold Base


!

Figure 11: Each Binding Strip column must be located directly above a PCR tube from the PCR strip.

➠ Pipette 100μl of preheated (70 °C) Buffer E directly onto the membrane of each Binding Strip using a single channel pipette. New tips have to be used for each sample.

➠ Incubate for 5 minutes at room temperature. ➠ Slowly apply the vacuum to a maximum of -400 mbar by opening the Manifold’s Valve. ➠ Quickly stop the vacuum by closing the Manifold’s Valve directly after all of the liquid has passed

through the columns to avoid foam formation. ➠ Switch off the Vacuum Pump and release the vacuum. ➠ Close the labelled PCR tubes for storage.

For optimal performance of isolated DNA in downstream applications we recommend elution with the supplied Buffer E. Store the extracted DNA at -20 °C.For the further analysis with assays from the oCheck® product line refer to the respective Instructions for Use (PapilloCheck® and PapilloCheck® high-risk).


33

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11. TROUBLESHOOTING

Problem and cause Comments and suggestions

NO OR POOR DNA YIELD

Incomplete lysis

Insufficientcellnumbers in sample.

Reagents not applied properly Suboptimal elution of DNA from the column

•Sample not thoroughly homogenised and mixed with Buffer L1 / Proteinase K.The mixture must be vigorously vortexed immediately after Buffer L1 addition.

•Decreased Proteinase K activity: Divide the prepared Proteinase K solution into aliquots and store at –20 °C for up to 6 month. Do not refreeze individual aliquots.

•Prepare Buffers L4, W2, Carrier RNA and Proteinase K solution according to section 8.1 or 10.1. Add ethanol to the lysates before loading them onto the columns.

•Preheat Buffer E to 70 °C before elution. Apply Buffer E directly onto the center of the silica membrane.

•Elution efficiencies decrease dramatically, if elution is done with buffers with pH < 7.0. Use slightly alkaline elution buffers such as Buffer E (pH 8.5).

COLUMNS CLOGGED

Incomplete lysis

Reagents not applied properly

•Sample not thoroughly homogenised and mixed with Buffer L1/Proteinase K. The mixture must be vigorously vortexed immediately following Buffer L1 addition.

•Decreased Proteinase K activity: Divide the prepared Proteinase K solution into aliquots and store at –20 °C for up to 6 months. Do not refreeze individual aliquots.

•Prepare Buffers L4, W2, Carrier RNA and Proteinase K solution according to section 8.1 or 10.1. Add ethanol to the lysates before loading them on the columns.

SUBOPTIMAL PERFORMANCE OF DNA IN ENZYMATIC REACTIONS

Carryover of ethanol or salt

Contamination of DNA with inhibitory substances

•Be certain to centrifuge at least 1 minute at 11,000 g in order to remove all of ethanolic Buffer W2 before DNA elution. If, for any reason, the level of Buffer W2 has reached the column outlet after drying, repeat the centrifugation.

•Do not chill Buffer W2 before use. Cold buffer will not remove salt effectively. Equilibrate Buffer W2 to room temperature before use.

•Do not elute DNA with TE buffer. EDTA may inhibit enzymatic reactions. Repurify DNA and elute in Buffer E.

•Cervical smear samples which are very dilute and show no visible cells should be concentrated to achieve a higher cell extraction yield. Centrifuge up to 1000 µl sample for 5 minutes at 11,000 g and resuspend the pellet in 250 µl of distilled water.

•Dilute an aliquot of the eluted DNA sample 1:5 before using it in the PCR reaction. Repeat the PCR reaction with the diluted sample DNA.

•If the A260/280 ratio of the eluate is below 1.6, repeat the purification procedure: add 1 volume Buffer L4 plus 1 volume ethanol (96-100%) to the eluate. Load the mixture onto a spin column and proceed with the DNA binding step (section 8.3.5 or 10.3.5) of the standard protocol.


34

12. TECHNICAL ASSISTANCE

Greiner Bio-One employs a Technical Service Department staffed with experienced scientists with extensive practical and theoretical expertise in molecular biology and oCheck® products.If you have any questions or experience any difficulties concerning oCheck® products, please do not hesitate to contact your local Greiner Bio-One distributor.

13. PERFORMANCE CHARACTERISTICS

The oCheck® DNA Extraction Kit is an IVD accessory to assays from the oCheck® product line. Analytical and specific performance of the oCheck® DNA Extraction Kit were tested in combination with the relevant assay. Please refer to the respective oCheck® products Instructions for Use for a detailed description of the performance [PapilloCheck® (Greiner Bio-One; Cat. No. 465 060), PapilloCheck® high-risk (Greiner Bio-One; Cat. No. 505 060) and PelvoCheck® CT/NG (Greiner Bio-One; Cat. No. 504 002)].


SHORT PROTOCOL

DNA Extraction

Room 114. SHORT PROTOCOLS

14.1 Short protocol for Single Column Preparation (Cat. No. 515 040)

DNA Extraction using oCheck® DNA Extraction Kit Single Column Preparation (Greiner Bio-One Cat. No. 515 040)

1. PREPARATION

➠ Check that the centrifuge is set to room temperature (18-25 °C) ➠ Prepare working solutions according to chapter 8.1 ➠ Set heat blocks or water baths to 56 °C and 70 °C ➠ Pre-heat elution Buffer E to 70 °C ➠ Prepare samples according to chapter 8.2 ➠ Pipet 250 μl of sample into a 1.5 or 2.0 ml reaction tube

2. PRE-LYSIS

➠ 80 µl Buffer L1 ➠ 2.4 μl Carrier RNA solution ➠ 20 μl Proteinase K solution ➠ Briefly vortex! ➠ 56 °C for at least 30 minutes ➠ Briefly centrifuge!

3. LYSIS

➠ 250 µl Buffer L4 ➠ Briefly vortex! ➠ 70 °C for 15 minutes ➠ Briefly centrifuge!

4. ADJUST DNA BINDING CONDITIONS

➠ 300 μl ethanol ➠ Briefly vortex ➠ Briefly centrifuge


SHORT PROTOCOL

DNA Extraction

Room 1

5. BIND DNA

➠ Prepare Spin Columns ➠ 450 μl lysate ➠ 1 minute at 11,000 g ➠ 450 μl lysate ➠ 1 minute at 11,000 g

6. WASH SPIN COLUMNS

➠ 500 μl Buffer W1 ➠ 1 minute at 11,000 g ➠ 600 μl Buffer W2 ➠ 1 minute at 11,000 g

7. DRY SPIN COLUMNS

➠ Centrifuge 1 minute at 11,000 g

8. DNA ELUTION

➠ 100 μl Buffer E (pre-heated 70 °C) ➠ 1 minute at room temperature (18-25 °C) ➠ 1 minute at 11,000 g


SHORT PROTOCOL

DNA Extraction

Room 114.2 Short protocol for 8 Columns Preparation (Cat. No. 515 050)

DNA Extraction using oCheck® DNA Extraction Kit - 8 Columns Preparation (Greiner Bio-One Cat. No. 515 050)

1. PREPARATION

➠ Check that the centrifuge is set to room temperature (18-25 °C) ➠ Prepare working solutions according to chapter 10.1 ➠ Set heat blocks or water baths to 56 °C and 70 °C ➠ Pre-heat elution Buffer E to 70 °C ➠ Prepare samples according to chapter 10.2 ➠ Pipet 250 μl of sample into a 1.5 or 2.0 ml reaction tube

2. PRE-LYSIS

➠ 80 µl Buffer L1 ➠ 2.4 μl Carrier RNA solution ➠ 20 μl Proteinase K solution ➠ Briefly vortex! ➠ 56 °C for at least 30 minutes ➠ Briefly centrifuge!

3. LYSIS

➠ 250 µl Buffer L4 ➠ Briefly vortex! ➠ 70 °C for 15 minutes ➠ Briefly centrifuge!

4. ADJUST DNA BINDING CONDITIONS

➠ 300 μl ethanol ➠ Briefly vortex ➠ Briefly centrifuge


SHORT PROTOCOL

DNA Extraction

Room 1

5. BIND DNA

➠ Pipet 900 μl lysate on the Binding Strips ➠ Apply vacuum: -200 mbar* ➠ Avoid foam formation!

6. WASH BINDING STRIPS

➠ 1st wash 600 μl Buffer W1 ➠ 2nd wash 900 µl Buffer W2 ➠ 3rd wash 900 µl Buffer W2

For each washing step: ➠ 30-60 seconds ➠ -200 mbar* ➠ Avoid foam formation!

7. DRY BINDING STRIPS

➠ 10 minutes at -600 mbar*

8. DNA ELUTION

➠ 100 μl Buffer E (pre-heated 70 °C) ➠ 5 minutes at room temperature (18-25 °C) ➠ Apply vacuum: -400 mbar* ➠ Avoid foam formation!

* Reduction of atmospheric pressure: Depending on sample viscosity extension of filtration time or increase of vacuum (e.g. -200 to – 400 mbar) may be required.

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