KingFisher Pure DNA Blood Kit Instruction Manual
Contents
1. Thermo Scientific KingFisher Pure DNA Blood Kit Revision 1 0 N14034 March 2013 hermo 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 PAR2. 96 plate B Tip 12 tip comb Empty C Empty Empty Empty D Empty Empty Empty E Empty Empty Empty F Wash 1_1 Wash Buffer 1 900 ul G Wash 1_2 Wash Buffer 1 800 ul H Wash 2 Wash Buffer 2 800 ul 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 Elution Buffer is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Elution Buffer 100 pl 4 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on a Blood DNA plate 5 Start the PURE_DNABlood_Duo protocol using the KingFisher Duo and load the plate and elution strip 22 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 Switch on the KingFisher Duo making sure that you are using the Thermo ScientificTM KingFisherTM Duo 12 pin magnet head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_DNABlood_Duo protocol Insert the Blood DNA 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 th
3. Pure DNA Blood 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 KingFisher instruments can be found in their respective user manuals The Bindlt Software 3 2 protocols optimized for the KingFisher Pure DNA Blood Kit are available for the KingFisher Flex 96 and 24 and KingFisher Duo For more information go to www thermoscientific com kingfisherinfo or contact your local authorized distributor Thermo Scientific KingFisher Pure DNA Blood Kit 11 Chapter 2 Product Description Table 2 1 Overview of KingFisher Flex and KingFisher Duo magnetic particle processors KingFisher Flex KingFisher Duo 96 formats 24 format 12 format 6 format ae 20 1000 200 5000 ui 30 1000 pi 200 5000 pi volume Capacity Up to 96 Up to 24 Upto 12 Up to 6 samples per samples per samples per samples per run sample run sample run sample run sample volume 200 ul volume 1 ml volume 200 ul volume 1 ml Magnetic head 96 inter 24 format for 12 pin magnet 6 pin magnet changeable KingFisher Flex head for head for formats for 24 deep well Microtiter deep KingFisher Flex PCR plate plate well 96 plate 24 deep well KingFisher Flex plate 96 KF plate Microtiter deep well 96 plate Plates KingFisher Flex KingFisher Flex Micr
4. binding between the purified DNA 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 DNA is released The volume of the Elution Buffer can be modified depending on user requirements concerning the purified DNA concentration The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of the buffer but this can slightly reduce the overall DNA 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 DNA Blood Kit 27 Chapter 6 General Information Table 6 1 Available elution volumes of the KingFisher Flex and KingFisher Duo Elution KingFisher instrument volumes KingFisher Flex with 96 deep well head elution in a KingFisher Flex 50 150 pl 96 KF plate KingFisher Flex with 96 deep well head elution in a Microtiter deep 50 1000 pul well 96 plate KingFisher Flex with 24 deep well head 200 5000 ul KingFisher Duo with 12 pin magnet head elution in an elution strip 30 130 pl KingFisher Duo with 12 pin magnet head elution in a Microtiter deep 50 1000 pul well 96 plate To maximize the yield of purified DNA avoid t
5. from combustible material Take off immediately all contaminated clothing This material and its container must be disposed of as hazardous waste 14 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Storage Conditions and Preparation of the Reagents Storage Conditions Upon arrival store the KingFisher Magnetic Beads at 4 C All the other buffers and reagents included in the KingFisher Pure DNA Blood Kit can be stored at room temperature 15 25 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 The reagents are stable for up to three years from the manufacturing date Preparation of the Binding Buffer and Wash Buffers Add 96 100 ethanol to the bottles containing Binding Buffer Wash Buffer 1 and Wash Buffer 2 as indicated below in Table 4 1 prior to the first use Table 4 1 Instructions for the preparation of the buffers Add the indicated volume per bottle 96 samples 384 samples Cat No 98010196 Cat No 98010496 Binding Wash Wash Binding Wash Wash Buffer Buffer1 Buffer2 Buffer Buffer 1 Buffer 2 Concentrated buffer 23 ml 100ml 40ml 90 ml 100 mi 40 ml Ethanol 96 100 23 ml 100 ml 200ml 90 ml 100 ml 200 ml Total volume 46 ml 200 ml 240 ml 180 mil 200 ml 240 ml After preparing each so
6. the Reagents 15 DOFAGE GONCIIONS aus oia Leas ees saws aS 15 Preparation of the Binding Buffer and Wash Buffers 15 Protocols and Pipetting Instructions 17 Handling of KingFisher Magnetic BeadS 17 Instructions for KingFisher Flex with 96 Deep Well Plates 17 Instructions for KingFisher Flex with 24 Deep Well Plates 19 Instructions for KingFisher Duo with 12 pin Magnet Head and 96 Deep Well Plates 0 0 0 0 c cece eects n eens 21 Instructions for KingFisher Duo with 6 pin Magnet Head and 24 Deep Well PIRES cocinan es dad 24 Quantification and Determination of the Purity of DNA 26 General Information 22000ceeeeeee eee eeees 27 Reagent Specificity and Volumes 0 00 cee eee 27 Handling of Magnetic Beads 0 eee o 27 Binding Wash and Elution Steps o0oooo o o 27 Decontamination and Disinfection of Sample Material 28 Troublesho0ting lt lt lt ooooommrommmmmmmmmmmmo 29 Ordering Information oooooooooororon 31 NOTE For more details on storing the kit reagents refer to Storage Conditions on page 6 Thermo Scientific KingFisher Pure DNA Blood Kit 3 Thermo Fisher Scientific Kit Content Table 1 1 Thermo Scientific KingFisher Pure DNA Blood Kit Cat No 98010196 98010496 Package size 96 samples 384 samples Proteinase K 2x1 ml 8x1ml L
7. 7003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 24 48 pcs deep well plate 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003520 KingFisher Duo elution strip 40 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well 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 Fisher Scientific Thermo Scientific KingFisher Pure DNA Blood Kit 7 Thermo Fisher Scientific Product Description Introduction The KingFisher Pure DNA Blood Kit is designed for rapid automated purifi cation of genomic DNA from whole blood samples using Thermo Scientific TM KingFisher instruments The DNA purified using the KingFisher Pure DNA Blood Kit is of high quality and free of proteins nucleases and other contaminants or inhibitors It is therefore suitable for direct use in many different downstream applications such as PCR polymerase chain reaction restriction analysis and several other enzymatic reactions Intended Use The KingFisher Pure DNA Blood Kit is developed for purification of DNA from whole blood samples using paramagnetic particles The reagents and specific plastic consumables are designed for use with the KingFisher Flex and KingFisher Du
8. Buffer to cover the KingFisher Magnetic Beads completely during the elution step Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of products from other manufacturers may cause unsuitable mixing and affect the yield of purified DNA Low purity Insufficient washing causes impurities in the Elution Buffer Blood clots in the sample cause impurities in the purified DNA Continued Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Blood Kit 29 Appendix A Troubleshooting Cont Possible cause and actions Magnetic particles remaining in the sample or elution well 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 Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the Elution Buffer 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 KingFisher Magnetic Beads that occasionally remain a
9. KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs Thermo Scientific KingFisher Pure DNA Blood Kit 31 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 pcs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 24 48 pcs deep well plate 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003520 KingFisher Duo elution strip 40 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well 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 32 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Notes Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Blood Kit 33 Notes 34 Thermo Scientific KingFisher Pure DNA Blood 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 cha
10. NA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield Instructions for KingFisher Duo with 12 pin Magnet Head and 96 Deep Well Plates These instructions are intended for DNA purification from 200 ul of blood using the KingFisher Pure DNA Blood Kit Cat No 98010196 or 98010496 and the KingFisher Duo with 96 deep well plates When using the KingFisher Pure DNA Blood Kit for the first time prepare the Binding Buffer 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 DNA Blood Kit 21 Chapter5 Protocols and Pipetting Instructions 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 KingFisher Duo elution strip 2 Prepare the Blood DNA 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 name and Row Row name Content ee Blood DNA A Sample Blood sample 200 ul plate Lysis Buffer 100 pl a Proteinase K 20 ul
11. TICULAR 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 ocio ir aa veces 5 Storage CONGIIONS 2006 e240 ges 8 o ee ee 6 Additional Reagents Required 0 cece eee eee eee 6 Product Description 000ccee cece cence eee eens 9 INFODUCHON iin NO 9 IO 2 00 rer 9 Principle and Procedure oooocccccocoo ooo 9 Kit SPOCHICATIONS o esi op os abe ke Gad ew ee ae ea 10 KingFisher Magnetic Particle Processors 10 Safety Information lt lt lt lt lt lt oooo0ooo lt oooooommo s 13 Storage Conditions and Preparation of
12. ance at 320 nm 280 nm and 260 nm The concentration of DNA can be defined with the absorbance at 260 nm A One unit at 260 nm corresponds to 50 g of DNA per ml The ratio between the A A indicates the purity of the DNA The value for 260 280 DNA should be gt 1 7 2 0 It is recommended that A correction is used for the absorbance values Subtract the A from the A and A ratios to remove the effects of carryover of the magnetic particles e Concentration of DNA sample 50 ug ml x A 060 Agog X dilution factor e Total amount of DNA isolated concentration x volume of sample in ml e Purity of DNA sample Aso Agog Ang A 280 200 26 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific 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 lt is strongly recommended that the volume of reagents in each well is kept within the limits specified in the KingFisher Flex User Manual or the 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 The amount of beads in the wells affects the yield of the purified DNA Binding Wash and Elution Steps The
13. ce the overall DNA yield Thermo Scientific KingFisher Pure DNA Blood Kit 23 Chapter5 Protocols and Pipetting Instructions Instructions for KingFisher Duo with 6 pin Magnet Head and 24 Deep Well Plates These instructions are intended for DNA purification from 1 ml of blood using the KingFisher Pure DNA Blood Kit Cat No 98010196 or 98010496 and the KingFisher Duo with 6 pin magnet head and 24 deep well plates When using the KingFisher Pure DNA Blood Kit for the first time prepare the Binding Buffer Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents 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 two empty Thermo Scientific KingFisher Flex 24 deep well plates 2 Prepare the Blood DNA plates 1 and 2 Add the following reagents to two KingFisher Flex 24 deep well plates Note that row B on the Blood DNA plate 1 is reserved for the tip comb and should be left empty Note that row C on the Blood DNA plate 1 and row B on the Blood DNA plate 2 are also left empty Plate name and type Blood DNA plate 1 KingFisher Flex 24 deep well plate Row Row name Content Blood sample Sample reagent volume 1000 ul Lysis Buffer 500 ul Proteinase K 60 u
14. dd the KingFisher Magnetic Beads and Binding Buffer to row A on the Blood DNA plate 1 during the dispense step When the KingFisher Duo pauses at the dispense step after the lysis step at approximately 5 minutes after starting the protocol run remove the Blood DNA plate 1 from the instrument and add the well suspended magnetic bead suspension and Binding Buffer supplemented with ethanol to row A on the Blood DNA plate 1 Plate name and type Row Rowname Content Reagent volume POUDA A Sample KingFisher 70 ul plate 1 Magnetic KingFisher Flex Beads 24 deep well Binding Buffer 1600 pl plate Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Place the Blood DNA plate 1 back into the instrument and press OK After the pause the protocol will continue to completion Thermo Scientific KingFisher Pure DNA Blood Kit 25 Chapter5 Protocols and Pipetting Instructions 8 When the protocol is completed remove the plates according to the instructions on the KingFisher Duo display and switch off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield Quantification and Determination of the Purity of DNA Itis recommended to measure the absorb
15. e KingFisher Duo is to be run as a standalone instrument transfer the PURE_DNABlood_Duo protocol to the KingFisher Duo 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 KingFisher Magnetic Beads and Binding Buffer supplemented with ethanol to row A during the dispense step When the KingFisher Duo pauses at the dispense step after the lysis step at approximately 5 minutes after starting the protocol run remove the plate from the instrument and add the well suspended magnetic bead suspension and Binding Buffer Supplemented with ethanol to row A on the Blood DNA plate Reagent volume Row name Content per well KingFisher Magnetic 25 pl Beads Binding Buffer 400 ul Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Place the Blood DNA 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 DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of buffer but this can slightly redu
16. eep Sample KingFisher 25 pul well 96 plate Magnetic Beads Binding Buffer 400 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 DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield Instructions for KingFisher Flex with 24 Deep Well Plates These instructions are intended for DNA purification from 1 ml of blood using the KingFisher Pure DNA Blood Kit Cat No 98010196 or 98010496 and the KingFisher Flex with 24 deep well plates Thermo Scientific KingFisher Pure DNA Blood Kit 19 Chapter5 Protocols and Pipetting Instructions When using the KingFisher Pure DNA Blood Kit for the first time prepare the Binding Buffer Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and
17. equilibrate to room temperature 15 25 0 1 Take six empty Thermo Scientific KingFisher Flex 24 deep well plates 2 Prepare the Sample plate Add the following reagents to the Sample plate and leave the plate at room temperature while the other plates are being filled BIS Plate type Plate name Content Sampie aden number volume per well KingFisher Flex Sample Blood sample 1000 ul 24 desp well Lysis Buffer 500 yl plate Proteinase K 60 pl 3 Fill the other buffer plates as follows Plate on Plate type Plate name Content Reagent volume 2 Wash 1_1 Wash Buffer 1 3700 ul 3 KingFisher Flex Wash 1_2 Wash Buffer 1 3700 ul 24 deep well 4 plate Wash 2 Wash Buffer 2 3700 yl 5 Elution Elution Buffer 500 ul 4 Place a Thermo Scientific KingFisher Flex 24 tip comb on a Tip Plate i e an empty KingFisher Flex 24 deep well plate 5 Start the PURE_DNABlood_Flex24 protocol using the KingFisher Flex 24 Switch on the KingFisher Flex making sure that you are using the Thermo Scientific KingFisher Flex 24 deep well head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Flex Start the PURE_DNABlood_Flex24 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 20 Thermo Scientific KingFishe
18. gents included in the KingFisher Pure DNA Blood Kit can be stored at room temperature 15 25 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 The reagents are stable for up to three years from the manufacturing date Additional Reagents Required e 96 100 ethanol EtOH molecular biology grade Table 1 2 Thermo Scientific KingFisher magnetic particle processors Cat No Product 5400100 KingFisher Duo magnetic particle processor 5400630 KingFisher Flex magnetic particle processor with 96 deep well head 5400640 KingFisher Flex magnetic particle processor with 24 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 97002610 KingFisher Flex 24 deep well tip comb and plate 50 pcs 97002540 KingFisher Flex 96 KF plate 200 pl 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 6 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Kit Content Chapter 1 Table 1 4 Thermo Scientific KingFisher Duo consumables Cat No Product Package size 9
19. he lowest permitted volumes of Elution Buffer in the KingFisher instruments The Elution Buffer should cover the KingFisher Magnetic Beads completely and any possible sedimented magnetic beads should be completely resuspended In addition the volume of the Elution Buffer should be adequate for efficient mixing of the beads in order to obtain a maximal release of the purified DNA 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 28 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Troubleshooting Problem Possible cause and actions Low DNA yield Make sure that ethanol was added to the Binding Buffer Wash Buffer 1 and Wash Buffer 2 before use Follow the instructions for Preparation of the Binding Buffer and Wash Buffers on page 15 Do not let the KingFisher Magnetic Beads dry as this may result in lower elution efficiency Efficient lysis of the blood cells increases the DNA yield Prolonged storage or repeated freeze thaw cycles of the sample material may reduce the DNA yield There should be an adequate volume of the Elution
20. ions refer to Chapter 4 Storage Conditions and Preparation of the Reagents Thermo Scientific KingFisher Pure DNA Blood Kit 17 Chapter5 Protocols and Pipetting Instructions 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 MicrotiterTM deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates 2 Prepare the Sample plate 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 on Plate type Plate name Content volume per well 1 Microtiter deep Sample Blood sample 200 pl well 96 plate Lysis Buffer 100 ul Proteinase K 20 ul 3 Fill the other plates as follows Plate Reagent volume ar Plate type Plate name Content per well 2 Wash 1_1 Wash Buffer 1 900 ul 3 Mlicrotiter deep Wash 1_2 Wash Buffer 1 800 ul well 96 plate 4 Wash 2 Wash Buffer 2 800 ul 5 KingFisher Flex Elution Elution Buffer 150 ul 96 KF plate 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_DNABlood_Flex96 protocol using the KingFisher Flex 96 and load the plates as in
21. l B Tip Empty Empty Empty Wash Buffer 1 Empty 3700 ul Plate name and type Blood DNA plate 2 KingFisher Flex 24 deep well plate Sample reagent Row name Content ne A Elution Elution Buffer 500 pul B Empty Empty Empty c Wash 2 Wash Buffer 2 3700 pl D Wash 1_2 Wash Buffer 1 3700 ul 24 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 3 Place a Thermo Scientific KingFisher Duo 6 tip comb into row B on the Blood DNA plate 1 4 Start the PURE_DNABlood_Duo1 ml protocol using the KingFisher Duo and load the plates Switch on the KingFisher Duo making sure that you are using the Thermo Scientific KingFisher Duo 6 pin magnet head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_DNABlood_Duo1ml protocol Insert the Blood DNA plates into the instrument as indicated on the KingFisher Duo display and press OK After all the plates have been loaded into the instrument the protocol will start When the KingFisher Duo is to be run as a standalone instrument transfer the PURE_DNABlood_Duo1ml protocol to the KingFisher Duo 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 A
22. lution mark the bottle to indicate that the step has been completed The buffers can be stored at room temperature Thermo Scientific KingFisher Pure DNA Blood Kit 15 Thermo Fisher Scientific Protocols and Pipetting Instructions Before beginning the DNA purification protocol carefully read through the Thermo Scientific KingFisher Flex User Manual Cat No NO7669 or the Thermo Scientific KingFisher Duo User Manual Cat No N12420 and the 7hermo Scientific Bindit Software for KingFisher Instruments version 3 2 User Manual Cat No NO7974 Bindit Software 3 2 protocols can be found 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 DNA purification from 200 ul of blood using the KingFisher Pure DNA Blood Kit Cat No 98010196 or 98010496 and the KingFisher Flex with 96 deep well plates When using the KingFisher Pure DNA Blood Kit for the first time prepare the Binding Buffer Wash Buffer 1 and Wash Buffer 2 For more instruct
23. nd 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 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 can be carried out manually or partially automatically using automatic 10 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Product Description Chapter 2 dispensers for example the Thermo Scientific MultidropTM 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 the developed protocol onto the onboard software and run it directly from the instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting in high purity DNA 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
24. nge 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
25. o magnetic particle processors as part of an integrated system The KingFisher Pure DNA Blood Kit enables extraction of DNA from fresh or frozen blood treated with EDTA ethylenediaminetetraacetic acid or citrate The KingFisher Pure DNA Blood Kit is only intended for research use not for clinical or diagnostic use The user is responsible for validating the performance of the Thermo Scientific KingFisher instrument and the KingFisher Pure DNA Blood Kit for any particular use as the performance of the kits has not been validated for any specific organism or downstream application Principle and Procedure The KingFisher Pure DNA Blood Kit uses magnetic particle technology for DNA purification The Thermo Scientific KingFisher technology combines the speed and efficiency of DNA purification with easy handling of magnetic Thermo Scientific KingFisher Pure DNA Blood Kit 9 Chapter2 Product Description particles The purification process requires no phenol chloroform extraction and needs very little hands on time The first step of the protocol lyses the blood cells after which the DNA can bind to the surface of the KingFisher Magnetic Beads in the presence of the Binding Buffer 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 DNA bound to the KingFisher Magnetic Beads is transferred thr
26. otiter deep KingFisher Flex 96 KF plate 24 deep well well 96 plate 24 deep well 20 200 ul plate 50 1000 ul plate 96 well PCR 200 5000 ul KingFisher Duo 200 5000 pul plate skirted elution strip 20 100 ul 30 130 ul Microtiter deep well 96 plate 60 1000 pul Tip combs KingFisher Flex KingFisher Flex KingFisher Duo KingFisher Duo 96 tip comb for 24 tip comb for 12 tip comb 6 tip comb PCR magnets deep well 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 DNA Blood Kit Thermo Fisher Scientific Safety Information The following components of the KingFisher Pure DNA Blood 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 chloride Harmful if swallowed Irritating to Polyethylene glycol octylphenol skin Risk of serious damage to ethe
27. ough the wash steps Two different Wash Buffers are used followed by an air drying step High quality DNA is eluted into the Elution Buffer and is ready for subsequent downstream processes Kit Specifications The KingFisher Pure DNA Blood Kit is designed for rapid automated preparation of highly pure DNA from whole blood using KingFisher magnetic particle processors If a dispense step requiring the addition of the Binding Buffer is excluded the approximate processing time is 30 minutes for the purification of 96 samples on the KingFisher Flex and 12 samples on the KingFisher Duo when the sample volume is 200 ul The obtained DNA can be used directly in various downstream applications Fresh or frozen blood treated with either EDTA or citrate can be used The procedure is optimized for sample volumes of 200 ul or 1 ml but the protocol can be scaled to sample volumes between these volumes Typically 3 9 jig of DNA can be purified from 200 pl of whole blood with an A A ratio of gt 1 7 2 0 The DNA yields depend on the sample type the sample storage and the number of white blood cells 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 a
28. r Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_DNABlood_Flex24 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 KingFisher Magnetic Beads and Binding Buffer to the Sample plate during the dispense step When the KingFisher Flex pauses at the dispense step after the lysis step at approximately 5 minutes after starting the protocol run remove the plate from the instrument and add the well suspended magnetic bead suspension and Binding Buffer supplemented with ethanol to the Sample plate Plate er Plate type Plate name Content Reagent volume 1 KingFisher Flex Sample KingFisher 70 ul 24 deep well Magnetic Beads plate Binding Buffer 1600 ul Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Place the Sample plates 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 DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final D
29. r eyes Harmful to aquatic organisms and may cause long term adverse effects in the aquatic environment 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 gloves and eye face protection This material and its container must be disposed of as hazardous waste Avoid release to the environment Refer to special instruction safety data sheets Continued Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Blood 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 In case of accident or if you feel unwell seek medical advice immediately This material and its container must be disposed of as hazardous waste Binding Buffer conc Sodium perchlorate Harmful if swallowed Explosive when mixed with combustible material Keep away from combustible material Do not breathe gas fumes vapor spray Take off immediately all contaminated clothing Wear suitable protective clothing This material and its container must be disposed of as hazardous waste Wash Buffer 1 conc Sodium perchlorate Explosive when mixed with combustible material Keep away
30. structed on the KingFisher Flex 96 instrument display 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_DNABlood_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 18 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_DNABlood_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 KingFisher Magnetic Beads and Binding Buffer supplemented with ethanol to the Sample plate during the dispense step When the KingFisher Flex pauses at the dispense step after the lysis step at approximately 5 minutes after starting the protocol run remove the plate from the instrument and add the well suspended magnetic bead suspension and Binding Buffer supplemented with ethanol to the Sample plate Plate Reagent volume ter Plate type Plate name Content per well 1 Microtiter d
31. ttached to the tip combs at the end of the process do not affect the DNA yield as the DNA has already been released from the KingFisher Magnetic Beads into the Elution Buffer Magnetic particles remaining in the sample or elution well If the KingFisher magnetic particle processor does not work properly refer to the relevant user manual of the KingFisher instrument in use Inhibition of downstream enzymatic reactions If the purified DNA contains residual salt use the correct order for the Wash Buffers Always wash the KingFisher Magnetic Beads with Wash Buffer 1 first and then proceed with Wash Buffer 2 30 Thermo Scientific KingFisher Pure DNA Blood Kit Thermo Fisher Scientific Thermo Fisher Scientific Ordering Information Table B 1 KingFisher Pure DNA Blood Kits Cat No Product Package size 98010196 KingFisher Pure DNA Blood Kit 96 98010496 KingFisher Pure DNA Blood 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
32. ysis Buffer 15 ml 60 ml KingFisher Magnetic Beads 2x 1 4 ml 10 6 ml Binding Buffer conc 23 ml 90 ml Wash Buffer 1 conc 100 ml 4x 100 ml Wash Buffer 2 conc 40 ml 2x40 ml Elution Buffer 30 ml 3x30 ml Addition of ethanol required The KingFisher Pure Blood DNA Kit Cat No 98010196 or 98010496 is intended for the purification of blood samples using the Thermo Scientific KingFisher Flex with a 96 deep well head or the Thermo Scientific KingFisher Duo with a 12 pin head and a sample volume of 200 ul The kit is also suitable for use with the KingFisher Flex with a 24 deep well head or the KingFisher Duo with a 6 pin head and a sample volume of 1 ml When a 1 ml sample volume is used the kit can handle approx 25 or 100 samples depending on the kit package size 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 Thermo Scientific KingFisher Pure DNA Blood Kit 5 Chapter 1 Kit Content 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 Storage Conditions Upon arrival store the Thermo Scientific KingFisher Magnetic Beads at 4 C All the other buffers and rea
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