KingFisher Pure Viral NA Kit Instruction Manual
Contents
1. Thermo Scientific KingFisher Pure Viral NA Kit Revision 1 0 N14040 March 2013 Copyright 2013 Thermo Fisher Scientific Inc All rights reserved Tween is a trademark of ICI Americas Inc Virkon is a trademark of E l du Pont de Nemours and Company or its affiliates All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Reproduction of the accompanying user documentation in whole or in part is prohibited Disclaimer Thermo Fisher Scientific reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without prior notice as part of continuous product development Although this manual has been prepared with every precaution to ensure accuracy Thermo Fisher Scientific assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information This instruction manual supersedes all previous editions Products are for Research Use Only Not for use in diagnostic procedures The Product will operate substantially in conformance with Thermo Fisher Scientific s published specifications THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES WHETHER EXPRESSED OR IMPLIED ORAL OR WRITTEN WITH RESPECT TO THE PRODUCTS INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF PRODUCT QUALITY CONDITION DESCRIPTION MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR2. Cat No 98070196 or 98070496 and the KingFisher Flex with 96 deep well plates When using the KingFisher Pure Viral NA Kit for the first time prepare the Carrier RNA Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents Thermo Scientific KingFisher Pure Viral NA Kit 17 Chapter 5 Protocols and Pipetting Instructions When beginning a new purification procedure prepare a fresh mixture of Carrier RNA and Lysis Buffer by adding 25 ul of Carrier RNA to 1 ml of Lysis Buffer Mix with a pipette or by vortexing Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 0 1 Take four empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates 2 Prepare the Sample plate i e a Microtiter deep well 96 plate Add the following reagents to the Sample plate and leave the plate at room temperature while the other plates are being filled Plate Sample reagent er Plate type Plate name Content volume per well 1 Microtiter deep Sample Sample 200 yl well 96 plate plasma serum or other Lysis Buffer 200 ul including the Carrier RNA Proteinase K 50 pl 3 Fill the other plates as follows Plate Reagent volume on Plate type
3. Plate name Content per well 2 Wash 1 Wash Buffer 1 900 ul 3 Microtiter deep Wash 2_1 Wash Buffer 2 800 ul well 96 plate 4 Wash 2_2 Wash Buffer 2 800 ul 5 KingFisher Flex Elution Nuclease free 100 ul 96 KF plate water 4 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate i e an empty KingFisher Flex 96 KF plate 5 Start the PURE_ViralNA_Flex96 protocol using the KingFisher Flex 96 and load the plates as instructed on the KingFisher Flex 96 instrument display 18 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 Switch on the KingFisher Flex making sure that you are using the Thermo Scientific KingFisher Flex 96 deep well head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Flex Start the PURE_ViralNA_Flex96 protocol Insert the Tip Plate and the filled plates into the instrument as indicated on the KingFisher Flex display After all the plates have been loaded into the instrument the protocol will start When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_ViralNA_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Binalt Software for KingFisher Instruments version 3 2 User Manual 6 Add the KingF
4. found in Chapter 4 Using the software in the Binalt Software for KingFisher Instruments version 3 2 User Manual 6 Add the KingFisher Magnetic Beads and 100 isopropanol to row A during the dispense step When the KingFisher Duo pauses at the dispense step after the lysis step at approximately 15 minutes after starting the protocol run remove the plate from the instrument and add the well suspended magnetic bead suspension and 100 isopropanol into row A on the Viral NA plate Plate name Content Reagent volume A Sample KingFisher Magnetic 25 ul Beads Isopropanol 450 pl Resuspend the KingFisher Magnetic Beads well by vortexing before use Thermo Scientific KingFisher Pure Viral NA Kit 21 Chapter 5 Protocols and Pipetting Instructions 7 Place the Viral NA plate back into the instrument and press OK After the pause the protocol will continue to completion 8 When the protocol is completed remove the plate and elution strip according to the instructions on the KingFisher Duo display and switch off the instrument Store the purified nucleic acids accordingly The purified nucleic acids are ready for use in downstream applications NOTE The final RNA concentration in the nuclease free water may increase if the purified NA is eluted into a smaller than recommended volume of water but this can slightly reduce the overall RNA yield Specific Instructions for Various Sample Materials When
5. manufacturing date Additional Reagents Required e 96 100 ethanol EtOH molecular biology grade e 100 isopropanol molecular biology grade Reagents required for specific sample pretreatments refer to Chapter 5 e 1xPBS pH 7 4 e 0 9 NaCl solution o TE buffer pH 8 0 e 0 5 MEDTA Table 1 2 Thermo KingFisher magnetic particle processors Cat No Product 5400100 KingFisher Duo magnetic particle processor 5400630 KingFisher Flex magnetic particle processor with 96 deep well head Table 1 3 Thermo Scientific KingFisher Flex consumables Cat No Product Package size 97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs 97002540 KingFisher Flex 96 KF plate 200 ul 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 6 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Kit Content Chapter 1 Table 1 4 Thermo Scientific KingFisher Duo consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003520 KingFisher Duo elution strip 40 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples Thermo Fishe
6. of the KingFisher instrument in use Inhibition of If the purified NA contains residual salt use the correct order for downstream the Wash Buffers Always wash the KingFisher Magnetic Beads enzymatic reactions with Wash Buffer 1 first and then proceed with Wash Buffer 2 28 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Ordering Information Table B 1 KingFisher Pure Viral NA Kits Cat No Product Package size 98070196 KingFisher Pure Viral NA Kit 96 98070496 KingFisher Pure Viral NA Kit 384 Table B 2 KingFisher Flex consumables Cat No Product Package size 97002514 KingFisher Flex 96 tip comb for PCR magnet 80 pcs 97002524 KingFisher Flex 96 tip comb for KF magnet 100 pcs 97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs 97002610 KingFisher Flex 24 deep well tip comb and plate 50 pcs 97002540 KingFisher Flex 96 KF plate 200 ul 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs Thermo Scientific KingFisher Pure Viral NA Kit 29 Appendix B Ordering Information Table B 3 KingFisher Duo consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 p
7. using the KingFisher Pure Viral NA Kit for the first time prepare the Carrier RNA Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents When beginning a new purification procedure prepare a fresh mixture of Carrier RNA and Lysis Buffer by adding 25 ul of Carrier RNA to 1 ml of Lysis Buffer Mix with a pipette or by vortexing Instructions for nucleic acid purification from 4 5 ml of urine These instructions are intended for nucleic acid purification from 4 5 ml aliquots of urine samples using the KingFisher Pure Viral NA Kit Cat No 98070196 or 98070496 and the KingFisher Flex or KingFisher Duo with 96 deep well plates 1 Add 500 ul of 0 5 M EDTA to 4500 ul of urine Urine samples may contain insoluble salt precipitates that can reduce nucleic acid yields thus limiting the sample volume used for purification 2 Centrifuge for 10 min at 800 x g Discard the supernatant 3 Resuspend the pellet in 200 ul of PBS or 0 9 NaCl and use as a sample in the purification 4 Proceed with the purification following the corresponding instructions for the KingFisher Flex or KingFisher Duo with 96 deep well plates for nucleic acid purification from 200 ul of cell free body fluid see page 17 or page 20 22 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5
8. your local authorized distributor Thermo Scientific KingFisher Pure Viral NA Kit 11 Chapter 2 Product Description Table 2 1 Overview of KingFisher Flex and KingFisher Duo magnetic particle processors KingFisher Flex 96 formats 24 format 12 format KingFisher Duo 6 format Processing volume 20 1000 pl 200 5000 pl 30 1000 ul 200 5000 ul Capacity Magnetic head Up to 96 samples per run sample volume 200 ul 96 inter changeable formats for PCR plate KingFisher Flex 96 KF plate Microtiter deep well 96 plate Up to 24 samples per run sample volume 1 ml 24 format for KingFisher Flex head for head for 24 deep well Microtiter deep KingFisher plate well 96 plate Flex 24 deep Up to 12 samples per run sample volume 200 ul 12 pin magnet Upto6 samples per run sample volume 1 ml 6 pin magnet well plate Plates KingFisher Flex KingFisher Flex Microtiter deep KingFisher 96 KF plate 24 deep well well 96 plate Flex 24 deep 20 200 ul 96 plate 50 1000 ul well plate well PCR plate 200 5000 ul KingFisher Duo 200 5000 ul skirted elution strip 20 100 yl 380 130 ul Microtiter deep well 96 plate 50 1000 ul Tip combs KingFisher Flex KingFisher Flex KingFisher Duo KingFisher 96 tip comb for 24 tip comb for 12 tip comb Duo 6 tip PCR magnets deep well c
9. 0 ul G Wash 2_1 Wash Buffer 2 800 ul H Wash 2_2 Wash Buffer 2 800 ul Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 3 Fill the KingFisher Duo elution strip as follows Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user and the nuclease free water is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Nuclease free water 100 pl 4 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on the Viral NA plate 5 Start the PURE_ViralNA_Duo protocol using the KingFisher Duo and load the plate and elution strip Switch on the KingFisher Duo making sure that you are using the Thermo Scientific KingFisher Duo 12 pin magnet head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_ViralNA_Duo protocol Insert the Viral NA plate and elution strip into the instrument as indicated on the KingFisher Duo display and press OK Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user When the KingFisher Duo is to be run as a standalone instrument transfer he PURE_ViralNA_Duo protocol to the KingFisher Duo The instructions for ransferring the protocol can be
10. 00 ml 200 ml Total volume 100 ml 250 ml 210 ml 250 ml After preparing each solution mark the bottle to indicate that the step has been completed The buffers can be stored at room temperature 16 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Before beginning the protocol for purification of nucleic acids carefully read hrough the 7hermo Scientific KingFisher Flex User Manual Cat No N07669 or the Thermo Scientific KingFisher Duo User Manual Cat No N12420 and the Thermo Scientific Bindlt Software for KingFisher Instruments version 3 2 User Manual Cat No NO7974 Bindlt Software protocols for the KingFisher Pure Viral NA Kit can be found in Bindlt Software 3 2 and at www thermoscientific com kingfisher Handling of KingFisher Magnetic Beads A homogeneous distribution of the KingFisher Magnetic Beads in the container is essential before the beads are transferred to the wells in order to ensure a high consistency between the wells To gain complete resuspension of the beads shake the container vigorously or vortex briefly The paramagnetic beads have a tendency to sediment relatively quickly Instructions for KingFisher Flex with 96 Deep Well Plates These instructions are intended for nucleic acid purification from 200 ul of plasma or serum samples using the KingFisher Pure Viral NA Kit
11. Determination of the Purity of Nucleic Acids The amount of viral nucleic acids in cell free body fluids is usually very small and the yields of purified nucleic acids are low In addition the Carrier RNA is copurified and affects the analyses of the yield with a spectrophotometer To determine the yield of viral nucleic acids it is recommended to use amplification techniques Thermo Scientific KingFisher Pure Viral NA Kit 23 Thermo Fisher Scientific General Information Reagent Specificity and Volumes A reagent must not be used with any kit other than that for which it is intended It is strongly recommended that the volume of reagents in each well or tube is kept within the limits specified in the KingFisher Flex User Manual or KingFisher Duo User Manualto avoid spillover and to maximize efficiency of performance Handling of Magnetic Beads The KingFisher Magnetic Beads should be mixed thoroughly before use to avoid the risk of transferring variable amounts of the beads to the respective wells or tubes The amount of beads in the wells or tubes affects the yield of the purified nucleic acids Binding Wash and Elution Steps The binding between the nucleic acids and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt The binding will remain throughout the wash steps until the elution where the nucleic acids are released The volume of the nuclease free water in the elution can be modified depending
12. Instructions for nucleic acid purification from buccal and nasal swabs These instructions are intended for nucleic acid purification from buccal and nasal swab samples using the KingFisher Pure Viral NA Kit Cat No 98070196 or 98070496 and the KingFisher Flex or KingFisher Duo with 96 deep well plates 1 To collect a swab sample scrape the swab 5 6 times against the inside of a cheek or nose 2 Swirl the swab for 2 3 min in 200 ul of PBS or 0 9 NaCl and use the solution as a sample in the purification 3 Proceed with the purification following the corresponding instructions for the KingFisher Flex or KingFisher Duo with 96 deep well plates for nucleic acid purification from 200 ul of cell free body fluid see page 17 or page 20 Instructions for nucleic acid purification from saliva These instructions are intended for nucleic acid purification from saliva samples using the KingFisher Pure Viral NA Kit Cat No 98070196 or 98070496 and the KingFisher Flex or KingFisher Duo with 96 deep well plates 1 Centrifuge the saliva sample for 5 min at 3 000 x g Discard the supernatant 2 Resuspend the pellet in 200 ul of PBS or TE buffer and use as a sample in the purification 3 Proceed with the purification following the corresponding instructions for the KingFisher Flex or KingFisher Duo with 96 deep well plates for nucleic acid purification from 200 ul of cell free body fluid see page 17 or page 20 Quantification and
13. PURPOSE THERMO FISHER SCIENTIFIC DOES NOT WARRANT THAT THE PRODUCTS ARE ERROR FREE OR WILL ACCOMPLISH ANY PARTICULAR RESULT THERMO FISHER SCIENTIFIC HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS Exclusion of Liability Thermo Fisher Scientific and its affiliates shall have no liability to an End User arising out of the use or inability to use the product including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise Thermo Fisher Scientific Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Appendix A Appendix B Table of Contents Kit Content i eweeter veces 5 Storage COnGIIONS cocoa ca ae riy 6 Additional Reagents Required 0 0 cece eee eee 6 Product Description 00ccee eee cence nn nn nn 9 INTOdUCUO socia matar indi id 9 INtENdE UsSb lt ooori io ee 9 Principle and Procedure 1 cece eects 10 Kit SPCHICATIONS o ese pecan does ee 10 KingFisher Magnetic Particle Processors 10 Safety Information lt lt lt lt lt lt oooo0ooo lt oooooommoos 13 Storage Conditions and Preparation of the Reag
14. can be carried out manually or partially automatically using automatic dispensers for example the Thermo Scientific Multidrop Combi and or the Thermo Scientific Versette Thermo Scientific Bindlt Software 3 2 can be used for running ready made and optimized protocols for the Thermo Scientific KingFisher Pure Kits It is also possible to transfer he developed protocol onto the onboard software and run it directly from he instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting n high purity nucleic acids without risk of carryover or cross contamination The KingFisher instrument family comprises four systems covering working volumes from 20 to 5000 ul Each system consists of an instrument specially designed plastic consumables and the easy to use Bindlt Software 3 2 The KingFisher Pure Viral NA Kit is optimized and ready for use with the KingFisher Flex or KingFisher Duo A ingFisher magnetic particle processors are intended for professional research se by trained personnel Detailed information and user instructions for the ingFisher instruments can be found in their respective user manuals As The Bindlt Software 3 2 protocols optimized for the KingFisher Pure Viral NA Kit are available for the KingFisher Flex 96 and KingFisher Duo For more information go to www thermoscientific com kingfisherinfo or contact
15. cs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 24 48 pcs deep well plate 97003520 KingFisher Duo elution strip 40 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 30 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific www thermoscientific com 2013 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details Thermo Fisher Scientific T 81 Wyman Street hermo Waltham MA 02451 SCIENTIFIC Part of Thermo Fisher Scientific
16. d be completely resuspended In addition the volume of the nuclease free water should be adequate for efficient mixing of the beads in order to obtain a maximal release of the purified nucleic acids from the beads Decontamination and Disinfection of Sample Material You should decontaminate the sample material and the reagents and plastics that have been in contact with the sample material in order to minimize the risk of contamination Use a decontaminant such as Virkon paying due attention to the manufacturer s instructions You should also take care of the appropriate treatment and or disposal of waste 26 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Troubleshooting Problem Possible cause and actions Low NA yield Make sure that the Carrier RNA was added to the Lysis Solution Make sure that isopropanol was added to Wash Buffer 1 and ethanol to Wash Buffer 2 before use Follow the instructions for Wash Buffer preparation on page 16 Do not let the KingFisher Magnetic Beads dry as this may result in lower elution efficiency Efficient Iysis of the samples increases the total NA yield Prolonged storage of the sample material may reduce the NA yield There should be an adequate volume of water to completely cover the KingFisher Magnetic Beads during the elution step Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of pr
17. ents 15 DOKAGS GONCIIONS cists nana ches REAR Ree AS 15 Preparation of the Carrier RNA cece ee 15 Preparation of the Lysis Buffer 0 c cece eee eee 15 Preparation of the Wash Buffers o oooooooo o o o 16 Protocols and Pipetting Instructions 17 Handling of KingFisher Magnetic BeadS 17 Instructions for KingFisher Flex with 96 Deep Well Plates 17 Instructions for KingFisher Duo with 96 Deep Well Plates 20 Specific Instructions for Various Sample Materials 22 Quantification and Determination of the Purity of Nucleic Acids 23 General Information 000cceeee eee nenn 25 Reagent Specificity and VoluMES o ooooooo 25 Handling of Magnetic Beads 0 cee eee 25 Binding Wash and Elution Steps 0 cece eee 25 Decontamination and Disinfection of Sample Material 26 Troubleshooting 00ceee cece cece cece eee eee 27 Ordering Information 00cceeee eee eee 29 NOTE For more details on storing the kit reagents refer to Storage Conditions on page 6 Thermo Scientific KingFisher Pure Viral NA Kit 3 Thermo Fisher Scientific Kit Content Table 1 1 Thermo Scientific KingFisher Pure Viral NA Kit Cat No 98070196 98070496 Package size 96 samples 384 samples Proteinase K 5 2 ml 21 ml Carrier RNA 1 vial 4 vials Lysis Buffer 40 ml 155 m
18. erformance of the kits has not been validated for any specific organism or downstream application Thermo Scientific KingFisher Pure Viral NA Kit 9 Chapter 2 Product Description Principle and Procedure The KingFisher Pure Viral NA Kit uses magnetic particle technology for viral RNA and DNA purification The Thermo Scientific KingFisher technology combines the speed and efficiency of nucleic acids purification with easy handling of magnetic particles The purification process requires no phenol chloroform extraction and needs very little hands on time The first step of the protocol lyses the sample after which the nucleic acids can bind to the surface of the KingFisher Magnetic Beads in the presence of isopropanol The KingFisher Magnetic Beads are highly reactive superparamagnetic beads The following three effective wash steps dispose of proteins cell debris and any residual contaminants while the nucleic acids bound to the KingFisher Magnetic Beads are transferred through the wash steps Two different Wash Buffers are used followed by an air drying step High quality nucleic acids are eluted into the nuclease free water and are ready for subsequent downstream processes Kit Specifications The KingFisher Pure Viral NA Kit is designed for rapid automated preparation of highly pure viral nucleic acids from body fluids and swab samples using KingFisher magnetic particle processors If a dispense step requiring the addi
19. her Scientific Thermo Scientific KingFisher Pure Viral NA Kit 13 Chapter 3 Safety Information Proteinase K Hazardous contents Proteinase Tritirachium album serine Cont Safety instructions May cause sensitization by inhalation Do not breathe gas fumes vapor spray Wear suitable protective clothing and gloves In case of accident or if you feel unwell seek medical advice immediately show the label where possible This material and its container must be disposed of as hazardous waste Wash Buffer 1 conc Guanidinium chloride Harmful if swallowed Irritating to eyes and skin Do not breathe gas fumes vapor spray In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste 14 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Storage Conditions and Preparation of the Reagents Storage Conditions Upon arrival of the kit store the KingFisher Magnetic Beads at 4 C and the Carrier RNA in the original aluminum bag at 20 C The Proteinase K solution is stable at room temperature until the seal of the vial is broken After being opened the vial should be stored at 20 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to th
20. isher Magnetic Beads and 100 isopropanol to the Sample plate during the dispense step When the KingFisher Flex pauses at the dispense step after the lysis step at approximately 15 minutes after starting the protocol run remove the plate from the instrument and add the well suspended magnetic bead suspension and 100 isopropanol to the Sample plate Plate Reagent volume ar Plate type Plate name Content per well 1 Microtiter deep Sample KingFisher 25 ul well 96 plate Magnetic Beads Isopropanol 450 ul Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Place the Sample plate back into the instrument and press Start After the pause the protocol will continue to completion 8 When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and switch off the instrument Store the purified nucleic acids accordingly The purified nucleic acids are ready for use in downstream applications NOTE The final RNA concentration in the nuclease free water may increase if the purified NA is eluted into a smaller than recommended volume of water but this can slightly reduce the overall RNA yield Thermo Scientific KingFisher Pure Viral NA Kit 19 Chapter 5 Protocols and Pipetting Instructions 20 Thermo Scientific KingFisher Instructions for KingFisher Duo with 96 Deep Well Plates These instructions are intended for nucleic acid purification from 200
21. l KingFisher Magnetic Beads 2x 1 4 ml 10 6 ml Wash Buffer 1 conc 55 ml 2x115 ml Wash Buffer 2 conc 50 ml 3x50 ml Nuclease free water 30 ml 2x30 ml Addition of ethanol or isopropanol required The KingFisher Pure Viral NA Kit Cat No 98070196 or 98070496 is intended for the purification of viral nucleic acids from body fluids and swab samples using the Thermo Scientific KingFisher Flex with a 96 deep well head or the Thermo Scientific KingFisher Duo with a 12 pin head The user will need the KingFisher Flex or KingFisher Duo magnetic particle processor for conducting purification Table 1 2 In addition several common laboratory instruments and consumables are required to conduct an efficient purification For more details refer to Chapter 5 Protocols and Pipetting Instructions Suitable consumables for the KingFisher Duo and KingFisher Flex are listed in Table 1 3 and Table 1 4 Thermo Scientific KingFisher Pure Viral NA Kit 5 Chapter 1 Kit Content Storage Conditions Upon arrival of the kit store the Thermo Scientific KingFisher Magnetic Beads at 4 C and the Carrier RNA in the original aluminum bag at 20 C The Proteinase K solution is stable at room temperature until the seal of the vial is broken After being opened the vial should be stored at 20 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to three years from the
22. oducts from other manufacturers may cause unsuitable mixing and affect the yield of purified NA Low purity Insufficient washing causes impurities in the eluted NA Continued Thermo Scientific KingFisher Pure Viral NA Kit 27 Appendix A Troubleshooting Cont Possible cause and actions Starting material that is too viscose prevents efficient collection of the KingFisher Magnetic Beads from the lysed sample The magnetic rods will not be able to collect all the particles unless the viscose samples are diluted before the beginning of the purification process Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the elution step the addition of a small amount of detergent e g Tween 20 may improve the results Alternatively centrifuge the eluates or place them on a magnet for a few minutes to collect the residual beads at the bottom of the well Carryover of the KingFisher Magnetic Beads does not affect most downstream processes Magnetic particles remaining in the sample or elution well KingFisher Magnetic Beads that occasionally remain attached to the tip combs at the end of the process do not affect the NA yield as the viral NA has already been released from the KingFisher Magnetic Beads into the eluate If the KingFisher magnetic particle processor does not work properly refer to the relevant user manual
23. omb KingFisher Flex magnets tip comb for KF magnets KingFisher Flex 96 tip comb for deep well magnets Heating Heating block temperature from Heating block temperature from temperature 5 C above ambient room 10 C to 75 C elution strip temperature to 115 C 4 C to 75 C at room temperature See the details above on the Plates row 12 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Safety Information The following components of the KingFisher Pure Viral NA Kit contain hazardous contents Table 3 1 Always wear a laboratory coat disposable gloves and goggles and follow the safety instructions provided in the kit instruction manual It is recommended that Good Laboratory Practice GLP is followed to guarantee reliable analyses Table 3 1 Safety precautions Reagent Hazardous contents Safety instructions Lysis Buffer Guanidium thiocyanate Harmful by inhalation in contact with skin and if swallowed Liberates very toxic gas In contact with acids Harmful to aquatic organisms and may cause long term adverse effects in the aquatic environment Keep the container in a well ventilated place Do not breathe gas fumes vapor spray Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste Avoid release to the environment Refer to special instructions safety data sheets Continued Thermo Fis
24. on user requirements concerning the purified nucleic acid concentration The final nucleic acid concentration in the nuclease free water may increase if the purified nucleic acids are eluted into a smaller than recommended volume of the buffer but this can slightly reduce the overall nucleic acid yield The modifications of the elution step must be done in Bindlt Software 3 2 and according to the volume ranges suitable for the KingFisher instrument The table below indicates the available elution volumes of the KingFisher instruments Thermo Scientific KingFisher Pure Viral NA Kit 25 Chapter 6 General Information Table 6 1 Available elution volumes of the KingFisher Flex and KingFisher Duo o A Elution KingFisher instrument omnes KingFisher Flex with 96 deep well head elution in a KingFisher Flex 96 50 150 pl KF plate KingFisher Flex with 96 deep well head elution in a Microtiter deep well 50 1000 pl 96 plate ingFisher Flex with 24 deep well head 200 5000 ul K KingFisher Duo with 12 pin magnet head elution in an elution strip 30 130 pl K ingFisher Duo with 12 pin magnet head elution in a Microtiter deep 50 1000 pl well 96 plate To maximize the yield of purified nucleic acids avoid the lowest permitted volumes of elution in the KingFisher instruments The nuclease free water in the elution should cover the KingFisher Magnetic Beads completely and any possible magnetic bead pellet s shoul
25. r Scientific Thermo Scientific KingFisher Pure Viral NA Kit 7 Thermo Fisher Scientific Product Description Introduction The KingFisher Pure Viral NA Kit is designed for rapid automated purification of viral nucleic acids from various samples such as plasma serum saliva and urine as well as from nasal buccal and urogenital swabs and blood samples using Thermo Scientific KingFisher instruments The nucleic acids NA purified using the KingFisher Pure Viral NA Kit are of high quality and free of proteins nucleases and other contaminants or inhibitors They are therefore suitable for direct use in many different downstream applications such as qPCR quantitative PCR RT qPCR reverse transcription qPCR and several other enzymatic reactions Intended Use The KingFisher Pure Viral NA Kit is developed for purification of viral nucleic acids from various samples such as plasma serum saliva and urine as well as from nasal buccal and urogenital swabs and blood samples using paramagnetic particles The reagents and specific plastic consumables are designed for use with the KingFisher Flex and KingFisher Duo magnetic particle processors as part of an integrated system The KingFisher Pure Viral NA Kit is only intended for research use not for clinical or diagnostic use The user is responsible for validating the performance of the KingFisher instrument and the KingFisher Pure Viral NA Kit for any particular use as the p
26. ree years from the manufacturing date Preparation of the Carrier RNA To prepare the Carrier RNA solution add 600 ul of nuclease free water to the vial of the Carrier RNA Incubate the freshly reconstituted Carrier RNA for 5 min at room temperature then mix thoroughly and briefly centrifuge the vial Use immediately or store in aliquots at 20 C Repeated freezing and thawing should be avoided Preparation of the Lysis Buffer Before each NA purification calculate the amount of Lysis Buffer needed For purification of one sample 200 ul of Lysis Buffer is required Prepare a fresh mixture of Carrier RNA and Lysis Buffer by adding 25 ul of Carrier RNA to 1 ml of Lysis Buffer Mix with a pipette or by vortexing Thermo Scientific KingFisher Pure Viral NA Kit 15 Chapter 4 Storage Conditions and Preparation of the Reagents Preparation of the Wash Buffers Add 100 isopropanol to the bottles containing Wash Buffer 1 and 96 100 ethanol to the bottles containing Wash Buffer 2 as indicated below in Table 4 1 prior to the first use Table 4 1 Instructions for the preparation of Wash Buffer 1 and Wash Buffer 2 Add the indicated volume of 100 isopropanol or 96 100 ethanol to each bottle 96 samples 384 samples Cat No 98070196 Cat No 98070496 Wash Buffer 1 Wash Buffer 2 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 55 ml 50 ml 115 ml 50 ml Isopropanol 100 45 ml 95 ml Ethanol 96 100 2
27. tion of isopropanol is excluded the approximate processing time is 40 minutes for the purification of 96 samples on the KingFisher Flex and 12 samples on the KingFisher Duo The obtained nucleic acids can be used directly in various downstream applications Suitable sample materials for purification are cell free body fluids such as serum plasma saliva and urine as well as nasal buccal and urogenital swabs and blood samples The sample type as well as the handling and storage of the sample affect the yield of the purified nucleic acids KingFisher Magnetic Particle Processors The KingFisher magnetic particle processors are designed for the automated transfer and processing of magnetic particles in microplate format The patented technology of the Thermo Scientific KingFisher systems is based on the use of magnetic rods covered with a disposable specially designed tip comb and plates or tubes Use only Thermo Scientific KingFisher plastic consumables as use of products from other manufacturers may cause unsuitable mixing or even instability in the KingFisher instrument 10 Thermo Scientific KingFisher Pure Viral NA Kit Thermo Fisher Scientific Thermo Fisher Scientific Product Description Chapter 2 The instrument functions without any dispensing or aspiration parts or devices Samples and reagents including magnetic particles are dispensed onto the plates according to the corresponding instructions Dispensing
28. ul of plasma or serum samples using the KingFisher Pure Viral NA Kit Cat No 98070196 or 98070496 and the KingFisher Duo with 96 deep well plates When using the KingFisher Pure Viral NA Kit for the first time prepare the Carrier RNA Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents When beginning a new purification procedure prepare a fresh mixture of Carrier RNA and Lysis Buffer by adding 25 ul of Carrier RNA to 1 ml of Lysis Buffer Mix with a pipette or by vortexing Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 C 1 Take one empty Microtiter deep well 96 plate and one Thermo Scientific TM KingFisher Duo elution strip 2 Prepare the Viral NA plate i e a Microtiter deep well 96 plate Add the following reagents to the rows Note that row B is reserved for the tip comb and should be left empty Note that rows C D and E are also left empty Plate name nen Content Sample reagent and type volume per well Viral NA plate A Sample Sample plasma 200 yl Microtiter deep serum or other well 96 plate Lysis Buffer 200 ul including the Carrier RNA Proteinase K 50 yl B Tip 12 tip comb Emp C Empty Empty Emp D Empty Empty Empty E Empty Empty Empty F Wash 1 Wash Buffer 1 90
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