instructions for use - analytik jena · introduction 4 . issue 04/2017 instant virus rna/dna kit ....

Instructions for UseLife Science Kits & Assays

AngruebeSchreibmaschinentextINSTANT Virus RNA/DNA Kit

AngruebeSchreibmaschinentext






This documentation describes the state at the time of publishing. It needs not necessarily agree with future versions. Subject to change!

Print-out and further use permitted with indication of source.© Copyright 2016, Analytik Jena AG, AJ Innuscreen GmbH

Manufacturer:AJ Innuscreen GmbHRobert-Rössle-Straße 1013125 BerlinMade in Germany!

Distribution/Publisher:Analytik Jena AGKonrad-Zuse-Straße 107745 Jena · Germany

Phone +49 3641 77 9400Fax +49 3641 77 [email protected]

Order No.:845-KS-4500010 10 reactions845-KS-4500050 50 reactions845-KS-4500250 250 reactions

Publication No.: HB_KS-1000_e_130315

Introduction

INSTANT Virus RNA/DNA Kit Issue 04/2017 1

Contents 1 Introduction ........................................................................................... 2

1.1 Intended use ................................................................................ 2 1.2 Notes on the use of this Instruction for Use (IFU) ..................... 4

2 Safety precautions ................................................................................. 5 3 Storage conditions ................................................................................ 6 4 Functional testing and technical assistance ......................................... 7 5 Product use and warranty ..................................................................... 8 6 Kit components ..................................................................................... 9 7 Components not included in the kit .................................................. 11 8 GHS classification ............................................................................... 12

8.1 Hazard phrases ......................................................................... 13 8.2 Precaution phrases ................................................................... 14 8.3 EU hazard statements .............................................................. 14

9 Recommended steps before starting ................................................ 15 10 General notes and safety recommendations on handling RNA ....... 16 11 General procedure for viral RNA/DNA extraction ............................. 18 12 Product specifications ........................................................................ 18 13 Protocol 1: Isolation of viral RNA/DNA from 400 μl of serum/plasma

using Extraction Tubes ....................................................................... 19 14 Protocol 2: Isolation of viral RNA/DNA from 400 μl of serum/plasma

using IC Spiking Tube ......................................................................... 21 Protocol 3: Isolation of viral RNA/DNA from 400 μl of serum/plasma using IC Spiking Tube (modified) ...................................................... 23

15 Troubleshooting ................................................................................. 25 16 Related Products ................................................................................ 26

Introduction

2 Issue 04/2017 INSTANT Virus RNA/DNA Kit

1 Introduction

1.1 Intended use

The INSTANT Virus RNA/DNA Kit has been designed for isolation both of viral RNA and DNA from different kinds of starting material. The extraction procedure is based on a new kind of chemistry (patent pending). The INSTANT Virus RNA/DNA Kit is optimized for the rapid preparation of highly pure viral RNA and DNA from cell free fluid biological samples, for example: plasma and serum and works with 400 μl liquid samples.

The procedure combines lysis of starting material with subsequent binding of viral RNA and DNA onto the surface of a Spin Filter membrane. After several washing steps the viral RNA and DNA is eluted from the membrane by using RNase-free water. Extraction chemistry and extraction protocol are optimized to get maximum of yield. Further, the kit should be used with corresponding detection assays, containing Carrier RNA and an Internal Extraction Control to monitor the purification, amplification, and detection processes (see related products).

Please note that the eluates contain both viral RNA/DNA and Carrier RNA. In case of using Carrier RNA the quantification of nucleic acids (isolated with this kit) by photometric or fluorometric methods is not possible. It is recommended to quantify extracted RNA/DNA with other methods like specific quantitative Real-time PCR.

The detection limit for certain viruses depends on the individual procedures, for example in-house PCR or commercial used detection assays.

The kit is intended for use by professional users. The kit has been designed to be used for a wide range of different downstream applications, like amplification reactions and further analytical procedures. Diagnostic results generated using the extraction procedure in conjunction with diagnostic tests should be interpreted with regard to other clinical or laboratory results. To reduce irregularities in diagnostic results, internal controls for downstream applications should be used.

Introduction


The internal control RNA/DNA can be used in combination with a corresponding RoboGene® Pathogen detection kit (Analytik Jena).

CONSULT INSTRUCTION FOR USE This package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of results cannot be guaranteed if there are any deviations from the instructions in this package insert.

Introduction


1.2 Notes on the use of this Instruction for Use (IFU)

For easy reference and orientation, the instructions for use (IFU) use the following warning and information symbols as well as the shown methodology:

Symbol Information

REF Catalogue number.

N Content Contains sufficient reagents for reactions.

Storage conditions Store at room temperature or shown conditions respectively.

Consult instructions for use This information must be observed to avoid improper use of the kit and the kit components.

Used by Expiry date.

Lot number The number of the kit charge.

CE-IVD symbol In vitro diagnostic medical device.

Manufactured by Contact information of manufacturer.

For single use only Do not use components twice.

Note / Attention Observe the notes marked in this way to ensure correct function of the device and to avoid operating errors for obtaining correct results.

The following systematic approach is introduced in the IFU:

The chapters and figures are numbered consecutively.

A cross reference is indicated with an arrow (e.g. “Notes on the use of this IFU” p. 4).

Working steps are numbered.

30 °C 15 °C

Safety precautions


2 Safety precautions

NOTE Read through this chapter carefully prior to guarantee your own safety and a trouble-free operation. Follow all the safety instructions explained in the IFU, as well as all messages and information, which are shown.

All due care and attention should be exercised in handling the materials and reagents contained in the kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact, flush eyes or skin with a large amount of water immediately.

FOR SINGLE USE ONLY! This kit is made for single use only!

ATTENTION! Don’t eat or drink components of the kit! The kit shall only be handled by educated staff in a laboratory environment!

If the buffer bottles are damaged or leaking, wear gloves and protective goggles when discarding the bottles in order to avoid any injuries. Analytik Jena AG has not tested the liquid waste generated during kit usage for potential residual infectious components. This case is highly unlikely, but cannot be excluded completely. Therefore, all liquid waste must be considered as potentially infectious and must be handled and discarded according to local safety regulations.

Clinical samples must always be considered as potentially infectious. Samples from risk patients must always be labeled and handled under consequent safety conditions. Please observe the federal, state and local safety and environmental regulations. Observe the usual precautions for applications using extracted nucleic acids. All materials and reagents used for RNA isolation should be free of RNases.

Storage conditions


ATTENTION! Do not add bleach or acidic components to the waste after sample preparation!

NOTE Emergency medical information in English and German can be obtained 24 hours a day from: Poison Information Center, Freiburg / Germany Phone: +49 (0)761 19 240.

For more information, please ask for the material safety data sheet (MSDS).

3 Storage conditions

The INSTANT Virus RNA/DNA Kit should be stored dry, at room temperature (15 °C–30 °C). When stored at room temperature the kit is stable until the expiration date printed on the kit label. Before every use make sure that all components have room temperature. If there are any precipitates within the provided solutions solve these precipitates by careful warming. For further information see chapter “Kit components“ p. 9.

Functional testing and technical assistance


4 Functional testing and technical assistance

The Analytik Jena AG guarantees the correct function of the kit for applications as described in the manual. The components of each INSTANT Virus RNA/DNA Kit were tested by isolation of the Internal Extraction Control and subsequent detection of IC RNA/DNA by Real-time RT-PCR, respectively.

We reserve the right to change or modify our products to enhance their performance and design. If you have any questions or problems regarding any aspects of the INSTANT Virus RNA/DNA Kit or other Analytik Jena AG products, please do not hesitate to contact us. For technical support or further information in Germany please dial +49 36 41 / 77 94 00. For other countries please contact your local distributor.

Product use and warranty


5 Product use and warranty

The kit is not designed for the usage of other starting materials or other amounts of starting materials than those, referred to in the intended use ( "Intended use" p. 2) and described in the summary ( "General procedure for viral RNA/DNA extraction" p. 18).

All plastic components and the chemistry are disposable products. When changing the starting material or the flow trace, no guarantee of the operability is issued. Since the performance characteristics of Analytik Jena AG kits have just been validated for the application described above, the user is responsible for the validation of the performance of Analytik Jena AG kits using other protocols then those described below. Analytik Jena AG kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the complete diagnostic system as required by CLIA’ 88 regulations in the U.S. or equivalent regulations required in other countries.

All products sold by the Analytik Jena AG are subjected to extensive quality control procedures and are warranted to perform as described when used correctly. Any problems should be reported immediately.

NOTE The kit is an in vitro diagnostic medical device!

Kit components


6 Kit components

10 50 250

AJ Innuscreen

845-KS-4500010 845-KS-4500050 845-KS-4500250

AJ Roboscreen

847-0259200601 847-0259200602 847-0259200603

Extraction Tubes/ IC Spiking Tube

Containing Carrier RNA and Internal Extraction Control. Delivered with the detection assays (→ "Related Products" p. 27).

Lysis Solution CLS

8 ml 30 ml 120 ml

Proteinase K for 1 x 1.5 ml working solution

for 4 x 1.5 ml working solution

for 14 x 1.5 ml working solution

Washing Solution HS

5 ml (final volume 10 ml)



Washing Solution LS



2 x 36 ml (final volume 180 ml)

RNase-free Water

2 ml 2 x 2 ml 25 ml

Spin Filter 10 50 5 x 50

Receiver Tubes (2.0 ml)

60 6 x 50 30 x 50

Elution Tubes (1.5 ml)

10 50 5 x 50

Instructions for Use

1 1 1

Kit components


10 50 250

AJ Innuscreen

845-KS-4500010 845-KS-4500050 845-KS-4500250

AJ

Roboscreen

847-0259200601 847-0259200602 847-0259200603

Initial steps Washing Solution HS Add 5 ml of 96-99.8 % ethanol to the bottle and mix thoroughly. Keep the bottle always firmly closed! Washing Solution LS Add 24 ml of 96-99.8 % ethanol to the bottle and mix thoroughly. Keep the bottle always firmly closed! Proteinase K Dissolve by addition of 1.5 ml of ddH2O, mix thoroughly and store as described below!

Washing Solution HS Add 15 ml of 96-99.8 % ethanol to the bottle and mix thoroughly. Keep the bottle always firmly closed! Washing Solution LS Add 64 ml of 96-99.8 % ethanol to the bottle and, mix thoroughly. Keep the bottle always firmly closed! Proteinase K Dissolve by addition of 1.5 ml of ddH2O, mix thoroughly and store as described below!

Washing Solution HS Add 70 ml of 96-99.8 % ethanol to the bottle and mix thoroughly. Keep the bottle always firmly closed! Washing Solution LS Add 144 ml of 96-99.8 % ethanol to the bottle and mix thoroughly. Keep the bottle always firmly closed! Proteinase K Dissolve by addition of 1.5 ml of ddH2O, mix thoroughly and store as described below!

Components not included in the kit


IMPORTANT Store lyophilized Proteinase K at 4 °C to 8 °C! Divide dissolved Proteinase K into aliquots and storage at -22 °C to -18 °C is recommended. Repeated freezing and thawing will reduce the activity dramatically!

STORAGE CONDITIONS All other components are stored at room temperature.

7 Components not included in the kit

1.5 ml tubes

2 ml tubes; optional

96–99.8 % ethanol

NOTE Use only absolute/pure ethanol, NO methylated or denatured alcohol! Isopropanol (molecular biology grade)

ddH2O for resuspension of Proteinase K

IMPORTANT NOTE For using the Internal Control please contact your local distributor!

30 °C 15 °C

GHS classification


8 GHS classification

Component Hazard contents GHS Symbol

Hazard phrases

Precaution phrases

EUH

Lysis Solution CLS

Guanidinium chloride2.5–

GHS classification


8.1 Hazard phrases

302 Harmful if swallowed.

314 Causes severe skin burns and eye damage.

315 Causes skin irritation.

317 May cause an allergic skin reaction.

319 Causes serious eye irritation.

332 Harmful if inhaled.

334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.

335 May cause respiratory irritation.

412 Harmful to aquatic life with long lasting effects.

GHS classification


8.2 Precaution phrases

101 If medical advice is needed, have product container or label at hand.

102 Keep out of reach of children.

103 Read label before use.

260 Do not breathe dust/fume/gas/mist/vapors/spray.

261 Avoid breathing dust/fume/gas/mist/vapors/spray

264 Wash thoroughly after handling.

280 Wear protective gloves/protective clothing/ eye protection/face protection.

310 Immediately call a POISON CENTER/doctor.

405 Store locked up.

501 Dispose of contents/container in accordance with local/regional/national/international regulations.

304+340 IF INHALED: Remove person to fresh air and keep comfortable for breathing.

303+361+ 353

IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water/shower.

305+351+ 338

IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

8.3 EU hazard statements

032 Contact with acids liberates very toxic gas

Short operation manual – INSTANT Virus RNA/DNA Kit | Publication No.: HB_KS-4500_e_101016 1

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INSTANT Virus RNA/DNA Kit

Protocol 1: Isolation of viral RNA/DNA from 400 µl of serum/plasma using Extraction Tube

Recommended steps before starting

Prepare Washing Solution HS, Washing Solution LS and Proteinase K according to the instruction

1. Starting material

Serum Plasma

Add 400 µl Lysis Solution CLS into an Extraction Tube

Add 400 µl sample and 40 µl Proteinase K to Extraction Tube

2. Lysis

Vortex: 10 seconds Incubation: 15 min @ 70 °C Centrifuge: shortly

3. Binding of RNA/DNA New Receiver Tubes

Add 800 µl Isopropanol Pipetting up and down Add Spin Filter to Receiver Tube Add 800 µl sample to Spin Filter 10,000 x g (∼12,000 rpm): 1 min Add Spin Filter to Receiver Tube Load residual sample 10,000 x g (∼12,000 rpm): 1 min

4. Washing New Receiver Tubes

Add 500 µl Washing Solution HS 10,000 x g (∼12,000 rpm): 1 min Add 650 µl Washing Solution LS 10,000 x g (∼12,000 rpm): 1 min Add 650 µl Washing Solution LS 10,000 x g (∼12,000 rpm): 1 min

5. Remove Ethanol New Receiver Tube

Discard filtrate Add Spin Filter to Receiver Tube Centrifuge: max speed, 5 min

2 Publication No.: HB_KS-4500_e_310317 | Short operation manual – INSTANT Virus RNA/DNA Kit

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6. Elution

Add Spin Filter to an Elution Tube Add preheated 60 µl RNase-free

water Incubation: 2 min @ RT 8,000 x g (∼10,000 rpm): 1 min Discard the Spin Filter Store Elution Tube on ice until

further use

Order No.: 847-0259200601

(845-KS-4500010)

10 reactions

847-0259200602

(845-KS-4500050)

50 reactions

847-0259200602

(845-KS-4500250)

250 reactions


Print-out and further use permitted with indication of source. © 2017 Analytik Jena AG, AJ Innuscreen GmbH

Analytik Jena AG Konrad-Zuse-Strasse 1 07745 Jena· Germany

Telefon +49 3641 77 70 Telefax +49 3641 77 9279 [email protected] www.analytik-jena.com

Manufacturer: AJ Innuscreen GmbH Robert-Rössle-Strasse 10 13125 Berlin


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Protocol 2: Isolation of viral RNA/DNA from 400 μl of serum/plasma using IC Spiking Tube


Prepare Washing Solution HS, Washing Solution LS, Proteinase K and IC Spiking Tube IC RNA according to the instruction


Serum Plasma

Vortex and centrifuge sample prior use.

Add 80 μl Proteinase K to an empty 2.0 ml Tube

Add 400 μl sample Mix well by pipetting up and

down Add 400 μl Lysis Solution CLS and

10 μl IC RNA solution

2. Lysis Vortex: 10 seconds Incubation: 10 min @ 70 °C Centrifuge: shortly


Add 800 μl Isopropanol Mix well by pipetting up and

down and centrifuge shortly Add Spin Filter to Receiver Tube Add 800 μl sample to Spin Filter 10,000 x g (12,000 rpm): 1 min Add Spin Filter to Receiver Tube Load residual sample 10,000 x g (12,000 rpm): 1 min


Add 500 μl Washing Solution HS 10,000 x g (12,000 rpm): 1 min Add 650 μl Washing Solution LS


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10,000 x g (12,000 rpm): 1 min Add 650 μl Washing Solution LS 10,000 x g (12,000 rpm): 1 min



6. Elution

Add Spin Filter to an Elution Tube Add preheated 60 μl RNase-free

water Incubation: 2 min @ 70°C 8,000 x g (10,000 rpm): 1 min Discard the Spin Filter Store Elution Tube on ice until

further use

Order No.: 847-0259200601

(845-KS-4500010)

10 reactions

847-0259200602

(845-KS-4500050)

50 reactions

847-0259200602

845-KS-4500250

250 reactions







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Protocol 3: Isolation of viral RNA/DNA from 400 μl of serum/plasma using IC Spiking Tube (modified)


Prepare Washing Solution HS, Washing Solution LS, Proteinase K and IC Spiking Tube IC RNA according to the instruction


Serum Plasma

Add 40 μl Proteinase K to an empty 2.0 ml Tube

Add 400 μl sample Vortex: 10 seconds Mix 10 μl IC solution and 400 μl

Lysis Solution CLS and add the Lysis/IC mix to the sample

2. Lysis Incubation: 15 min @ 70°C Centrifuge: shortly


Add 800 μl Isopropanol Vortex: 10 seconds Add Spin Filter to Receiver Tube Add 800 μl sample to Spin Filter 10,000 x g (12,000 rpm): 1 min Add Spin Filter to Receiver Tube Load residual sample 10,000 x g (12,000 rpm): 1 min


Add 500 μl Washing Solution HS 10,000 x g (12,000 rpm): 1 min Add 650 μl Washing Solution LS 10,000 x g (12,000 rpm): 1 min Add 650 μl Washing Solution LS 10,000 x g (12,000 rpm): 1 min


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6. Elution

Add Spin Filter to an Elution Tube Add preheated 60 μl RNase-free

water Incubation: 2 min @ RT 8,000 x g (10,000 rpm): 1 min Discard the Spin Filter Store Elution Tube on ice until

further use

Order No.: 847-0259200601

(845-KS-4500010)

10 reactions

847-0259200602

(845-KS-4500050)

50 reactions

847-0259200603

845-KS-4500250

250 reactions








9 Recommended steps before starting

Heat thermal mixer or water bath to 70 °C.

Ensure that the Washing Solution HS, Washing Solution LS and Proteinase K have been prepared according to the instruction ( "Kit components" p. 9).

Preheat aliquots of the RNase-free Water at 70 °C according to the desired elution volumes.

NOTE Do not use preheated ddH2O for Proteinase K! Centrifugation steps should be carried out at room temperature.

Avoid freezing and thawing of starting material.

General notes and safety recommendations on handling RNA


10 General notes and safety recommendations on handling RNA

RNA is far less stable than DNA. It is very sensitive to degradation by endogenous RNases in the biological material and exogenous RNases which are permanently present everywhere in the lab. To achieve satisfactory qualitative and quantitative results in RNA preparations, contaminations with exogenous RNases have to be reduced to a minimum in accordance with the following recommendations:

Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contaminations from surface of the skin or from dusty laboratory equipment.

Change gloves frequently and keep tubes closed.

Keep isolated RNA on ice.

Reduce preparation time as much as possible.

Use only sterile, disposable polypropylene tubes throughout the procedure (these tubes are generally RNase-free.)

Non-disposable plastic ware should be treated before use to ensure that it is RNase-free. Plastic ware should be thoroughly rinsed with 0.1 M NaOH, 1 mM EDTA followed by RNase-free water. You can also take chloroform-resistant plastic ware rinsed with chloroform to inactivate RNases.

All glassware should be treated before use to ensure that it is RNase-free. Glassware should be cleaned with detergent, thoroughly rinsed and oven baked at 240 °C for four or more hours before use. Autoclaving alone will not inactivate many RNases completely. Oven baking inactivates RNases and ensures that no other nucleic acids (such as plasmid DNA) are present on the surface of the glassware. You can also clean glassware with 0.1 % DEPC (diethyl pyrocarbonate). The glassware has to be immersed in 0.1 % DEPC solution for 12 hours at 37 °C and has to be autoclaved or heated to 100 °C for 15 min subsequently to remove residual DEPC.

Electrophoresis tanks should be cleaned with detergent solution (e.g. 0.5 % SDS), thoroughly rinsed with RNase-free water, rinsed with ethanol and finally allowed to dry.

General notes and safety recommendations on handling RNA


All buffers have to be prepared with DEPC-treated RNase-free ddH2O.

Avoid handling bacterial cultures, cell cultures or other biological sources of RNases in the same lab where the RNA purification will be performed.

Do not use equipment, glassware and plastic ware employed for other applications which might introduce RNase contaminations in the RNA isolation.

General procedure for viral RNA/DNA extraction


11 General procedure for viral RNA/DNA extraction

Lysis of starting material Binding of viral RNA and DNA on Spin Filter Washing of the bound RNA and DNA Elution of RNA and DNA

12 Product specifications

1. Starting material:

Serum and plasma (400 μl).

2. Time for isolation:

Approximately 30 minutes.

Protocol 1: Isolation of viral RNA/DNA from 400 μl of serum/plasma using Extraction Tubes


13 Protocol 1: Isolation of viral RNA/DNA from 400 μl of serum/plasma using Extraction Tubes

NOTE! Pre-fill the needed amount of RNase-free Water into a 2.0 ml reaction tube and incubate the RNase-free Water at 70 °C until the elution step.

1. Vortex and centrifuge the sample prior to use.

2. Open the Extraction Tube and add 400 μl Lysis Solution CLS. Add 400 μl of the sample and 40 μl of Proteinase K, mix vigorously by pulsed vortexing for 10 seconds.

3. Incubate the mixture for 15 min at 70 °C. After lysis centrifuge the extraction Tube shortly to remove condensate from the lid of the tube.

NOTE! We recommend to use a shaking platform for a continuous shaking of the sample. Alternatively, vortex the sample 3-4 times during incubation. No shaking will reduce the lysis efficiency.

4. Add 800 μl Isopropanol (not included in the kit) to the lysed sample, mix well by pipetting up and down several times or vortex shortly.

NOTE! It is important that the sample and the Isopropanol are mixed vigorously to get a homogeneous solution.

5. Apply 800 μl of the sample to the Spin Filter located in a 2.0 ml Receiver Tube. Close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.

Protocol 1: Isolation of viral RNA/DNA from 400 μl of serum/plasma using Extraction Tubes


NOTE! If the solution has not completely passed through the Spin Filter, centrifuge again at higher speed or prolong the centrifugation time.

6. Apply the residual sample to the Spin Filter located in a 2.0 ml Receiver Tube. Close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.

NOTE!

If the solution has not completely passed through the Spin Filter, centrifuge again at higher speed or prolong the centrifugation time. 7. Open the Spin Filter and add 500 μl Washing Solution HS, close the

cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.

8. Open the Spin Filter and add 650 μl Washing Solution LS, close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.


10. Centrifuge at max. speed for 5 minutes to remove all traces of ethanol. Discard the 2.0 ml Receiver Tube.

11. Place the Spin Filter into a 1.5 ml Elution Tube. Carefully open the cap of the Spin Filter and add 60 μl RNase-free water (alternatively 2 x 30 μl for improvement of yield) preheated to 70 °C. Close the cap and incubate at room temperature for 2 minutes. Centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Discard the Spin Filter, close the Elution Tube and place it on ice until further use.



14 Protocol 2: Isolation of viral RNA/DNA from 400 μl of serum/plasma using IC Spiking Tube



2. Open a 2.0 ml tube (not provided) and add 80 μl Proteinase K. Add 400 μl of the sample and mix well by pipetting up and down.

3. Add 400 μl Lysis Solution CLS and 10 μl IC solution from the IC Spiking Tube IC RNA; mix vigorously by pulsed vortexing for 10 seconds. Incubate the mixture at 70 °C for 10 minutes. After lysis centrifuge the 2.0 ml tube shortly to remove condensate from the lid of the tube.

NOTE! We recommend to use a shaking platform for a continuous shaking of the sample. Alternatively, vortex the sample 3-4 times during incubation. No shaking will reduce the lysis efficiency. 4. Add 800 μl Isopropanol (not included in the kit) to the lysed sample,

mix well by pipetting up and down several times or vortex shortly. Centrifuge shortly.


5. Apply 850 μl of the sample to the Spin Filter located in a 2.0 ml Receiver Tube. Close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.






7. Open the Spin Filter and add 500 μl Washing Solution HS, close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.




11. Place the Spin Filter into a 1.5 ml Elution Tube. Carefully open the cap of the Spin Filter and add 60 μl RNase-free water preheated to 70 °C. Close the cap and incubate at 70°C for 2 minutes. Centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Discard the Spin Filter, close the Elution Tube and place it on ice until further use.






2. Add 40 μl of Proteinase K to an empty 2.0 ml tube (not provided). Add 400 μl of the sample and mix vigorously by pulsed vortexing for 10 seconds.

3. Mix 10 μl IC solution from the IC Spiking Tube IC RNA with 400 μl Lysis Solution CLS. Add the mixture to the sample containing 2.0 ml tube (not provided). Incubate the mixture at 70 °C for 15 minutes. After lysis centrifuge the 2.0 ml tube shortly to remove condensate from the lid of the tube.

NOTE! We recommend to use a shaking platform for a continuous shaking of the sample. Alternatively, vortex the sample 3-4 times during incubation. No shaking will reduce the lysis efficiency.

4. Add 800 μl Isopropanol (not included in the kit) to the lysed sample, mix well by pipetting up and down several times or vortex shortly.


5. Apply 800 μl of the sample to the Spin Filter located in a 2.0 ml Receiver Tube. Close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.





NOTE!

If the solution has not completely passed through the Spin Filter, centrifuge again at higher speed or prolong the centrifugation time.

7. Open the Spin Filter and add 500 μl Washing Solution HS, close the cap and centrifuge at 10,000 x g (12,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new 2.0 ml Receiver Tube.




11. Place the Spin Filter into a 1.5 ml Elution Tube. Carefully open the cap of the Spin Filter and add 60 μl RNase-free Water (preheat to 70 °C). Close the cap and incubate at room temperature for 2 minutes. Centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Discard the Spin Filter, close the Elution Tube and place it on ice until further use.

Troubleshooting


15 Troubleshooting

Problem / probable cause Comments and suggestions

Clogged Spin Filter

Insufficient lysis and/or too much starting material

Increase lysis time. Increase centrifugation speed. Reduce amount of starting material.

Low amount of extracted viral RNA/DNA

Insufficient lysis

Increase lysis time. Reduce amount of starting material. Overloading of Spin Filter reduces yield!

Incomplete elution

Prolong the incubation time with RNase-free water to 5 minutes or repeat elution step once again. Take a higher volume of RNase-free water.

Insufficient mixing with Isopropanol Mix sample with Isopropanol by pipetting or by vortexing prior to transfer of the sample onto the Spin Filter.

Low concentration of extracted viral RNA

Too much RNase-free water Elute the viral RNA with lower volume of RNase-free water.

No Extraction Tubes are used Use Extraction Tubes for extraction of the sample, as described in the manual above.

Related Products


16 Related Products

Name Amount Order No.

Detection Assays

RoboGene HDV RNA Quantification Kit 2.0 66/72 rxn 847-02074005X4

132/136 rxn

847-02074005X2

RoboGene HBV DNA Quantification Kit 2.0 66/72 rxn 847-02075001X4

132/136 rxn

847-02075001X2

RoboGene® HCV RNA Quantification Kit 3.0 32 rxn 847-0207610032

96 rxn 847-0207610096

192 rxn 847-0207610192

engl. /1 – Analytik Jena AG, Jena

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Headquarters

Analytik Jena AGKonrad-Zuse-Str. 107745 Jena · Germany

Phone +49 3641 77 70Fax +49 3641 77 [email protected]

Pictures: Analytik Jena AGSubject to changes in design and scope of delivery as well as further technical development!

16-09-08_DRUCK_Manual_engl_mit CE-IVD_komplett.pdfEindruck_1.Seite_DB_englEindruck_2.Seite Innen_engl_mit CE-IVDEindruck_3.Seite_Innen_engl14-10-14_Eindruck_2.Seite Innen_engl_mit IVD.pdfSeite 1

14-11-20_Deckblatt_neue Positionierung.pdfEindruck_1.Seite_DB_englEindruck_2.Seite Innen_engl_ohne CE-IVDEindruck_3.Seite_Innen_engl

SH_KS-4500_CE_IVD_e_P1_310317.pdfINSTANT Virus RNA/DNA KitProtocol 1: Isolation of viral RNA/DNA from 400 µl of serum/plasma using Extraction Tube

16-09-08_WEB_Manual_engl_mit CE-IVD_komplett.pdfWEB_Manual_engl_RS.pdfWEB_Manual_engl_ohne CE_DB_RS_Innen+Aussen.pdfHB_BP-5100_e_110207 3HB_BP-5100_e_110207 4

14-10-14_WEB_Eindruck_2.Seite Innen_deu_mit CE-IVD.pdfSeite 1

SH_KS-4500_CE_IVD_e_P1_310317.pdfINSTANT Virus RNA/DNA KitProtocol 1: Isolation of viral RNA/DNA from 400 µl of serum/plasma using Extraction Tube

instructions for use - analytik jena · introduction 4 . issue 04/2017 instant virus rna/dna kit ....

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