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KingFisher Cell and Tissue DNA Kit

1. Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Rev 1 2 Cat no N11997 Copyright 2012 Thermo Fisher Scientific Inc First edition published in 2010 All rights reserved Reproduction of the accompanying user documentation in whole or in part is prohibited BindIt KingFisher Microtiter Multidrop NanoDrop and Versette are registered trademarks of Thermo Fisher Scientific All other trademarks and registered trademarks are the property of their respective holders 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 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 PARTICULAR PURPOS
2. C to 75 C in to 115 C room temperature 14 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Product Description KingFisher magnetic particle processors Table 2 1 Overview of Thermo Scientific KingFisher systems KingFisher mL KingFisher Processing volume 50 1000 pl 20 200 ul Capacity Up to 15 samples per run lysed sample Up to 24 samples per run volume 225 ul with the KingFisher Cell and Tissue DNA Kit Magnet heads 15 format 24 format Plates KingFisher mL tube special tube strip with KingFisher plate 100 or 200 pl 20 100 ul 1x 5 tubes 50 1000 pl or 20 200 ul Tip combs KingFisher mL tip comb 1 x 5 format KingFisher tip comb 1 x 12 format Heating No heating available No heating available temperature The BindIt Software 3 2 protocols optimized for the KingFisher Cell and Tissue DNA Kit are available for the KingFisher Flex 96 the KingFisher Duo and the KingFisher mL instruments BindIt Software 3 2 protocols for the Thermo Scientific KingFisher and the Thermo Scientific KingFisher 96 are available on request For more information contact your local authorized distributor Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 15 Product Description KingFisher magnetic particle processors 16 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Chapter 3
3. an empty KingFisher Flex 96 KF plate 5 Start the KF_TissueDNA_Flex96 protocol with the KingFisher Flex 96 and load the plates Switch on the KingFisher Flex and make sure that you are using the KingFisher Flex 96 deep well head and heating block Connect the PC with BindIt Software 3 2 to the KingFisher Flex Start the KF_TissueDNA_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 begin When the KingFisher Flex is to be run as a standalone instrument transfer the KF_TissueDNA_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the BindIt Software for KingFisher Instruments version 3 2 User Manual 24 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for DNA purification from 225 ul of lysed cell or tissue samples 6 After the run is completed remove the plates and store the purified DNA When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and turn off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications The final DNA conce
4. refer to Preparation of the or tissue samples Proteinase K working solution on page 19 1 Lyse the samples according to the instructions given in Lysing of samples on page 21 Perform the RNase A treatment optional The instructions for the treatment are given in RNase A treatment on page 22 2 Take four empty Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates 3 Prepare four Microtiter deep well 96 plates and one KingFisher Flex 96 KF plate as indicated in the table below Resuspend the KingFisher Magnetic Beads well e g by vortexing before transferring them from the bottle Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 23 Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for DNA purification from 225 ul of lysed cell or tissue samples Plate Plate Plate Content Reagent volume number type name per well 1 Microtiter Sample Lysed sample 225 ul deep well 96 plate KingFisher 25 pl Magnetic Beads Binding Buffer 360 pl 2 Microtiter Wash1 Wash Buffer 1 600 pl deep well 96 plate 3 Microtiter Wash2 Wash Buffer2 600 pl deep well 96 plate 4 Microtiter Wash3 Wash Buffer 3 800 pl deep well 96 plate 5 KingFisher Elution Elution Buffer 150 pl Flex 96 KF plate 4 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate that is
5. Cat No N12420 or the Thermo Scientific KingFisher mL User Manual Cat No 1508260 and the Thermo Scientific Bindlt Software for KingFisher Instruments version 3 2 User Manual Cat No N07974 BindIt Software 3 2 protocols for the KingFisher and the KingFisher 96 can be obtained on request A homogeneous distribution of the KingFisher Magnetic Beads in the container is essential before the beads are transferred to the wells or tubes in order to ensure a high consistency between the wells or tubes To gain complete resuspension of the beads shake the container vigorously or vortex briefly The KingFisher Magnetic Beads have a tendency to sediment relatively quickly in the Binding Buffer Once a premixture of the beads and the Binding Buffer has been made the mixture should be used immediately to avoid the risk of transferring variable amounts of the beads to the respective wells or tubes Use a maximum of 1 x 107 of cultured cells 1 ml of bacteria cultured overnight or 20 mg of tissue per sample Efficient lysis of the sample is an essential step before DNA purification in order to gain a good yield of high quality DNA The lysis step must disrupt the Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 21 Protocols and Pipetting Instructions Lysing of samples structures of the sample material completely in order to ensure a high yield of DNA Lysing of cells To lyse the cells centrifuge the sample ap
6. Safety Information The following components of the KingFisher Cell and Tissue DNA Kit contain hazardous contents Table 3 1 Wear a laboratory coat disposable gloves and goggles and follow the safety instructions given 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 Binding Buffer Hazardous contents Sodium perchlorate lt 20 and ethanol lt 50 Safety instructions Flammable Harmful if swallowed Keep away from food drink and animal feed Do not inhale dust Immediately remove all contaminated clothing Wash Buffer 1 Sodium perchlorate lt 15 and ethanol lt 24 Flammable Harmful if swallowed Keep away from food drink and animal feed Do not inhale dust Immediately remove all contaminated clothing Wash Buffer 2 Sodium perchlorate lt 15 and ethanol lt 24 Flammable Harmful if swallowed Keep away from food drink and animal feed Do not inhale dust Immediately remove all contaminated clothing Proteinase K Thermo Fisher Scientific Lyophilized Proteinase K Irritating to eyes respiratory system and skin May cause sensitization by inhalation Do not inhale dust Avoid contact with skin In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves Thermo Scientific Kin
7. 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 The samples can for example be diluted into 1 x PBS 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 0 02 Tween 20 may improve the results Carryover of the KingFisher Magnetic Beads to the Elution Buffer may affect the A A ratio Refer to Low purity on page 37 KingFisher Magnetic Beads that occasionally remain attached 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 If the KingFisher magnetic particle processor does not work properly refer to the relevant user manual of the KingFisher instrument in use 38 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Appendix B Ordering Information Table B 1 Thermo Scientific KingFisher Cell and Tissue DNA Kits Cat No Product Package size 97030196 KingFisher Cell and Tissue DNA Kit 1x 96 Table B 2 Thermo Scientific 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 f
8. combs 97003520 KingFisher Duo elution strip 40 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 96 plate 1 box tips combs plates and elution strips for 96 samples Table 1 5 Thermo Scientific KingFisher mL consumables Cat No Product Package size 97002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 20 x 45 pcs 97002131 KingFisher mL combi tubes and tip combs for 60 samples 60 97002141 KingFisher mL combi tubes and tip combs for 240 samples 240 Table 1 6 Thermo Scientific KingFisher consumables Cat No Product Package size 97002070 KingFisher tip comb 50 pcs 97002080 KingFisher plate 100 pl 50 pcs 97002084 KingFisher plate 200 pl 50 pcs 97002090 KingFisher plastics 100 pl 8 pack 8 plates and 8 tip combs 1 box 97002094 KingFisher plastics 200 pl 8 pack 8 plates and 8 tip combs 1 box Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 9 Kit Content 10 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Introduction Intended use Principle and procedure Thermo Fisher Scientific Chapter 2 Product Description The KingFisher Cell and Tissue DNA Kit is designed for rapid automated purification of DNA from cell or bacterial cultures and tissues using KingFisher instruments DNA purified using the KingFisher Cell and Tissue DNA Kit is of high quality and free of protein
9. instrument The instrument functions without any dispensing or aspiration parts or devices Samples and reagents including magnetic particles are dispensed into the plates according to the corresponding instructions Dispensing can be done manually or partially automatically using automatic dispensers for example the Thermo Scientific Multidrop Combi and or the Thermo Scientific Versette Thermo Scientific BindIt Software 3 2 can be used for running ready made optimized protocols for the Thermo Scientific KingFisher Kits It is also possible to transfer the defined protocol onto the onboard software and run it directly from the instrument The KingFisher instruments offer a rapid and automated solution for complicated and time consuming purification processes without risk of carryover or cross contamination resulting in high purity DNA 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 BindIt Software 3 2 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 13 Product Description KingFisher magnetic particle processors The KingFisher Cell and Tissue DNA Kit is optimized and ready for use with KingFisher systems The KingFisher magnetic particle processors are intended for professional research use by trained personnel Detailed information and user instructio
10. see below RNase A treatment RNase A treatment is recommended for the samples containing large amounts of RNA or if RNA disturbs downstream applications Add RNase A not included to the lysed sample at a final concentration of 1 8 mg ml in order to reduce the amount of RNA in the sample 22 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for DNA purification from 225 ul of lysed cell or tissue samples Incubation should be performed in accordance with the enzyme manufacturer s instructions To clear the lysate from debris centrifuge the samples for 5 minutes 6000 x g Transfer the supernatant to a new tube a Thermo Scientific Microtiter deep well 96 plate or a Thermo Scientific KingFisher mL tube strip and begin the purification of DNA using the KingFisher Flex or the KingFisher mL See the detailed instructions below Instructions for These instructions are for the DNA purification from KingFisher Flex 225 pl of lysed cell or tissue samples using the KingFisher with 96 deep well Cell and Tissue Kit Cat No 97030196 1 x 96 and the p KingFisher Flex with Microtiter deep well 96 plates plates for DNA purification from When using the KingFisher Cell and Tissue DNA Kit for 225 ul of d cell the first time prepare the working solution of Proteinase HI of lysed ce K For more instructions
11. then sedimented after a short centrifugation step The cleared lysates are transferred to Thermo Scientific KingFisher plates for processing with a Thermo Scientific KingFisher magnetic particle processor DNA binds to the surface of the Thermo Scientific KingFisher Magnetic Beads in the presence of a chaotropic salt The following effective wash steps dispose of proteins cell debris and any residual contaminants while the DNA bound to the KingFisher Magnetic Beads is transferred through the wash steps Three different Wash Buffers are used Although purified DNA is only rinsed rapidly in the Wash Buffer 3 this step considerably improves the purity of the DNA High quality DNA is eluted into the Elution Buffer and is ready for subsequent downstream processes such as enzymatic reactions Heat incubation is included in the elution step when using the KingFisher Flex or KingFisher Duo Kit specifications The KingFisher Cell and Tissue DNA Kit is designed for rapid automated preparation of highly pure DNA from cells and tissues using Thermo Scientific KingFisher magnetic particle processors The approximate processing time is 25 minutes for the purification of 96 samples in the KingFisher Flex and 12 samples in the KingFisher Duo or for the purification of 15 samples in the KingFisher mL The obtained DNA can be used directly in various downstream applications Suitable sample materials for the KingFisher Cell and Tissue DNA Kit are up to 20 m
12. tube strips and store the purified DNA When the protocol is completed remove the tubes and turn off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications To increase the DNA yield the Elution Buffer can be prewarmed to 55 C and dispensed into the Elution tubes during an additional pause step in the protocol before the elution step The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall DNA yield Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 31 Protocols and Pipetting Instructions Quantification and determination of the purity of DNA Summary Of Table 5 3 Summary of tube contents tube contents Tube Tube name Content Sample reagent volume A Sample Lysed sample 225 ul KingFisher 25 ul Magnetic Beads Binding Buffer 360 pl B Wash 1 Wash Buffer 1 600 ul C Wash 2 Wash Buffer 2 600 pl D Wash 3 Wash Buffer 3 800 pl E Elution Elution Buffer 150 pl Quantification and 1t is recommended to measure the absorbance at 320 nm determination of 280 nm and 260 nm The concentration of DNA can be P defined with the absorbance at 260 nm A3260 One unit the purity of DNA at 260 nm corresponds to 50 pg of DNA per ml The ratio between the A260 A280 indicates the purity of the DNA and the value for DNA
13. yield of the purified DNA Binding and The binding between the DNA and the KingFisher wash steps Magnetic Beads is strong in the presence of a chaotropic salt but chaotropic salts are not present in the Wash Buffer 3 and accordingly the binding is weak Avoid strong mixing speeds and releasing the beads into the Wash Buffer 3 in order to minimize the loss of DNA during the wash step A short wash and a slow mixing speed without releasing the beads into the buffer are recommended Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 33 General Information Elution step Elution step Carryover of ethanol to the Elution Buffer causes impurities in the Elution Buffer and may affect some downstream applications To remove traces of ethanol make sure that there is a wash step e g washing without releasing the beads or the drying step before the elution step is long enough There is a delicate balance between complete removal of the ethanol and loss of DNA The volume of the Elution Buffer can be modified depending on the 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 volume of the buffer but this can slightly reduce the overall DNA yield The modifications of the elution step must be done in BindIt Software 3 2 and according to the volume ranges suitable for the KingFis
14. C Empty Empty Empty D Empty Empty Empty E Empty Empty Empty F Wash 1 Wash Buffer 1 600 pl G Wash 2 Wash Buffer 2 600 pl H Wash 3 Wash Buffer 3 800 pl Elution strip Elution Elution Buffer 100 pl Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 29 Protocols and Pipetting Instructions Instructions for KingFisher mL for DNA purification from 225 ul of lysed cell or tissue samples Instructions for KingFisher mL for DNA purification from 225 ul of lysed cell or tissue samples These instructions are for the DNA purification from 225 ul of lysed cell or tissue samples using the KingFisher Cell and Tissue Kit Cat No 97030196 and the KingFisher mL When using the KingFisher Cell and Tissue DNA Kit for the first time prepare the working solution of Proteinase K For more instructions refer to Preparation of the Proteinase K working solution on page 19 A tube strip tray in the KingFisher mL can contain up to 15 separate KingFisher mL tube strips and one sample processing uses one tube strip with five tubes The orientation of the tube strip is fixed Note that the tube strips have to be positioned so that the slip ends are facing left One tip comb with five tips is used for processing five samples at a time 1 Lyse the samples according to the instructions given in Lysing of samples on page 21 Perform the RNase A treatment optional The instructions for the t
15. E 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 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 Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Thermo Fisher Scientific Kit COMPO naa cconcscoccsecctstcessssescseectcsnsetinnenecnenstnebsnusincbsneetaneesnsdeerbanstes 7 Product Description s scsscsscsessecseeseeseseesesseesnesteeneaeentens 11 latrod ction misa ei ee t deneubdoredosen tioned test 11 Intended sei miN a E A EA ERS 11 Principle and procedure s ssssssesssssssssssssesresresresresressessesresses 11 Kit sp cifications sonrie aana a aE EEEE 12 KingFisher magnetic particle processors sssssssssssssrssssesreessees 13 Safety Information ssssssssssssssssssessessesessesssssessessesssesseseesaeees 17 Storage Conditions and Prep
16. Empty E Empty Empty Empty F Wash 1 Wash Buffer 1 600 pl G Wash 2 Wash Buffer 2 600 pl H Wash 3 Wash Buffer 3 800 pl 4 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 5 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on a Cell and Tissue DNA plate 6 Start the KF_TissueDNA_Duo protocol with the KingFisher Duo and load the plate and elution strip Switch on the KingFisher Duo and make sure that you are using the KingFisher Duo 12 pin magnet head and heating block Connect the PC with BindIt Software 3 2 to the KingFisher Duo Start the KF_TissueDNA_ Duo protocol Insert the Cell and Tissue DNA plate and elution strip into the instrument as indicated on Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 27 Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 ul lysed cell or tissue samples 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 us
17. aration of the Working Solution s sssssssssssssssssssssssssssssssssssessesseseesscsaees 19 Storage conditons dss ennir an aaea i ann 19 Preparation of the Proteinase K working solution 0 0064 19 Protocols and Pipetting Instructions eeseeee Handling of KingFisher Magnetic Beads Eysing df Sam ples denero reS E AE EENS Lysine cells e e a EA Lysing of tissue samples nastean n RNase A treatment ccceecceececeessseseesessessssesessssesesseeeeees Instructions for KingFisher Flex with 96 deep well plates for DNA purification from 225 ul of lysed cell or tissue samples 23 Summary of plate contents 0 cece es seesesesteeeeeeeenees 25 Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 ul lysed cell or tissue Sam plese cei wecetececuseserereresersratereds dh conereiaverersus ters 26 Summary of plate and elution strip contents eee 29 Instructions for KingFisher mL for DNA purification from 225 ul of lysed cell or tissue samples Summary of tube contents Quantification and determination of the purity of DNA 32 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 5 Contents Chapter 6 Appendix A Appendix B General Information ssssssssssecsssssssssesstecstesseeseestesseesseenseeass 33 Reagent specificity and volumes cceeeeeeeeeeeeeeeeeeeeeeeeeeees 33 Handling of mag
18. ates When using the KingFisher Cell and Tissue DNA Kit for the first time prepare the working solution of Proteinase K For more instructions refer to Preparation of the Proteinase K working solution on page 19 1 Lyse the samples according to the instructions given in Lysing of samples on page 21 Perform the RNase A treatment optional The instructions for the treatment are given in RNase A treatment on page 22 2 Take one empty Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip 3 Prepare the Cell and Tissue DNA plate Microtiter deep well 96 plate Add the following reagents to the rows see next page Note that row B is reserved for the tip comb and should be left empty Note that rows C D and E are left empty Resuspend the KingFisher Magnetic Beads well e g by vortexing before removing them from the bottle 26 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 ul lysed cell or tissue samples Plate name and type Row Row name Content Reagent Sample volume per well Cell and Tissue DNA plate A Sample Lysed sample 225 ul Microtiter deep well KingFisher 25 pl 96 plate Magnetic Beads Binding Buffer 360 pl B Tip 12 tip comb Empty C Empty Empty Empty D Empty Empty
19. crease the yield by approximately 15 20 When using the KingFisher instrument without a heating option the Elution Buffer can be prewarmed to 55 C and dispensed into the Elution tubes or wells during an additional pause in the protocol before the elution step If some KingFisher Magnetic Beads remain in the Elution Buffer centrifuge the Elution plate briefly or place it on a magnet for a few minutes to collect the residual beads at the bottom of the well and transfer the supernatant to a new tube RNase A treatment is recommended if the purity of the DNA is not sufficient for downstream applications with the normal protocol Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 35 General Information Elution step 36 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Table A 1 Troubleshooting guide Problem Low DNA yield Appendix A Troubleshooting Possible cause and actions There should be an adequate volume of the Elution Buffer to cover the KingFisher Magnetic Beads completely during the elution step Do not let the KingFisher Magnetic Beads dry as this may result in lower elution efficiency Heating during the elution step or using a preheated Elution Buffer enhances the release of the DNA from the KingFisher Magnetic Beads and the yield of the purified DNA Efficient lysis of the cells or tissue increases the DNA yield P
20. er When the KingFisher Duo is to be run as a standalone instrument transfer the KF_TissueDNA_Duo protocol to the KingFisher Duo The instructions for transferring the protocol can be found in the BindIt Software for KingFisher Instruments version 3 2 User Manual 7 After the run is completed remove the plate and store the purified DNA When the protocol is completed remove the plate and elution strip according to the instructions on the KingFisher Duo display and turn off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall DNA yield 28 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Summary of plate and elution strip contents Plate name and type Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 ul lysed cell or tissue samples Table 5 2 Summary of plate and elution strip contents Row Row name Content Reagent Sample volume per well Cell and Tissue DNA plate A Sample Lysed sample 225 ul Microtiter deep well KingFisher 25 pl 96 plate Magnetic Beads Binding Buffer 360 pl B Tip 12 tip comb Empty
21. er tip comb 50 pcs 97002080 KingFisher plate 100 ul 50 pcs 97002084 KingFisher plate 200 ul 50 pcs 97002090 KingFisher plastics 100 pl 8 pack 8 plates and 8 tip combs 1 box 97002094 KingFisher plastics 200 pl 8 pack 8 plates and 8 tip combs 1 box 40 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Notes Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 41 Notes 42 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Thermo Fisher Scientific Oy Ratastie 2 P O Box 100 Fl 01621 Vantaa Finland www thermoscientific com N11997
22. g of tissue up to 1 x 107 of cells or up to 1 ml of bacteria cultured overnight It is possible to purify up to 20 ug of DNA depending on the sample and obtain an Ay o Azgo ratio of gt 1 6 1 9 per sample The yields of acquired purified DNA depend on the sample type and the method of sample storage The KingFisher Cell and Tissue DNA Kit can be processed at room temperature excluding the lysis step 12 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific KingFisher magnetic particle processors Thermo Fisher Scientific Product Description KingFisher magnetic particle processors with Proteinase K treatment which occurs outside the KingFisher instrument and requires heating Additionally heating of the elution step may increase the DNA yield by approximately 15 20 The KingFisher Magnetic Beads are highly reactive superparamagnetic beads The binding capacity is 0 4 ug of DNA per 1 ul of bead suspension 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 Use only Thermo Scientific KingFisher plastic consumables Use of products from other manufacturers may cause unsuitable mixing or even instability in the KingFisher
23. gFisher Cell and Tissue DNA Kit Instruction Manual 17 Safety Information 18 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Storage conditions Preparation of the Proteinase K working solution Thermo Fisher Scientific Chapter 4 Storage Conditions and Preparation of the Working Solution All buffers and reagents included in the KingFisher Cell and Tissue Kit can be stored at room temperature 20 25 C and are stable for up to one year from the manufacturing date The buffers are ready for use To prepare the Proteinase K working solution for the KingFisher Cell and Tissue DNA Kit add 2 6 ml of Proteinase K Buffer to the vial of the lyophilized Proteinase K The working solution should be stored at 20 C in aliquots Repeated freezing and thawing should be avoided Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 19 Storage Conditions and Preparation of the Working Solution Preparation of the Proteinase K working solution 20 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Handling of KingFisher Magnetic Beads Lysing of samples Thermo Fisher Scientific Chapter 5 Protocols and Pipetting Instructions Before beginning the DNA purification protocol carefully read through the Thermo Scientific KingFisher Flex User Manual Cat No N07669 the Thermo Scientific KingFisher Duo User Manual
24. her instrument The table below indicates the available elution volumes of the KingFisher instruments Table 6 1 Available elution volumes of Thermo Scientific KingFisher instruments KingFisher instrument Elution volume KingFisher 20 200 pl KingFisher mL 50 1000 ul KingFisher Duo with 12 pin magnet head 30 130 pl KingFisher Duo with 6 pin magnet head 200 5000 pl KingFisher Flex with 96 deep well head 50 150 pl elution in a KingFisher Flex 96 KF plate KingFisher Flex with 96 deep well head 50 1000 ul elution in a Microtiter deep well 96 plate KingFisher Flex with 96 well head 20 250 pl elution in a KingFisher Flex 96 KF plate KingFisher Flex with 24 deep well head 200 5000 pl 34 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific General Information Elution step To gain a maximal yield of purified DNA avoid the lowest permitted volumes of Elution Buffer in the KingFisher instruments The Elution Buffer should cover the KingFisher Magnetic Beads completely and any possible magnetic bead pellet 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 Elution can be conducted at room temperature The elution at 72 C in the KingFisher Flex and KingFisher Duo or at 55 C outside the KingFisher instrument will however in
25. netic beads oo eseeeeeeeeeeeeteeeeeeeeeees 33 Binding and wash steps ccecceseseseseeeeeeeeeeeeeseeeeeeeeeeeeeeeees 33 Elution Sepia a Goon ia 34 Troubleshooting sssssscsesscssssssssessessesesssaessssesaessesseatsseaeeessesees 37 Ordering Information sssssssssssssssssssessessesaesssssessseseeseeees 39 6 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Thermo Fisher Scientific Chapter 1 Kit Content Table 1 1 Thermo Scientific KingFisher Cell and Tissue DNA Kit Item KingFisher Cell and Tissue DNA Kit 1 x 96 Cat No 97030196 Package size 1x 96 samples KingFisher Magnetic Beads 2 6 ml Proteinase K 1 via Proteinase K Buffer 3 6 ml Lysis Buffer 25m Binding Buffer 40m Wash Buffer 1 65m Wash Buffer 2 65m Wash Buffer 3 100 ml Elution Buffer 20 m The KingFisher Cell and Tissue DNA Kit Cat No 97030196 is intended for the purification of cell and tissue samples using the Thermo Scientific KingFisher Flex with a 96 deep well head or the Thermo Scientific KingFisher Duo with a 12 pin head or Thermo Scientific KingFisher mL and a lysed sample volume of 225 ul Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 7 Kit Content Equipment and reagents to be supplied by the user e RNase A optional e Magnetic particle processor Table 1 2 Thermo Scientific KingFisher magnetic par
26. ns for the KingFisher instruments can be found in their respective user manuals Table 2 1 Overview of Thermo Scientific KingFisher systems KingFisher Flex KingFisher Duo 96 format 24 format 12 format 6 format Processing volume 20 1000 pl 200 5000 pl 30 1000 pl 200 5000 pl Capacity Up to 96 samples per Up to 24 samples Up to 12 samples Up to 6 samples run lysed sample per run per run per run volume 225 pl with the KingFisher Cell and Tissue DNA Kit Magnet heads 96 interchangeable 24 format for 12 pin magnethead 6 pin magnet head formats for PCR KingFisher 24deep for Microtiter deep for KingFisher Flex plate KingFisher well plate well 96 plate 24 deep well plate Flex 96 KF plate Microtiter deep well 96 plate Plates KingFisher Flex 96 KingFisher Flex 24 Microtiter deep well KingFisher Flex 24 KF plate 20 200 pl deep well plate 96 plate 50 1000 pl deep well plate Microtiter deep well 200 5000 ul KingFisher Duo 200 5000 ul 96 plate 50 1000 ul elution strip 96 well PCR plate 30 130 ul skirted 20 100 ul Tip combs KingFisher Flex 96 KingFisher Flex 24 KingFisher Duo KingFisher Duo tip comb for PCR tip comb for deep 12 tip comb 6 tip comb magnets KingFisher well magnets Flex tip comb for KF magnets KingFisher Flex 96 tip comb for deep well magnets Heating Heating block temperature from 5 C Heating block temperature from 10 C temperature above ambient room temperature to 75 C elution strip 4
27. ntration in the Elution Buffer may increase if the purified DNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall DNA yield Summary of Table 5 1 Summary of plate contents plate contents Plate Plate Plate number type name 1 Microtiter Sample deep well 96 plate Content Lysed sample KingFisher Magnetic Beads Binding Buffer Sample reagent volume per well 225 ul 25 pl 360 ul 2 Microtiter Wash 1 deep well 96 plate Wash Buffer 1 600 pl 3 Microtiter Wash 2 deep well 96 plate Wash Buffer 2 600 pl 4 Microtiter Wash 3 deep well 96 plate Wash Buffer 3 800 ul 5 KingFisher Elution Flex 96 KF plate Elution Buffer 150 pl 6 KingFisher Tip Plate Flex 96 KF plate Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 25 Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 ul lysed cell or tissue samples Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for DNA purification of 225 pl lysed cell or tissue samples These instructions are for the DNA purification from 225 ul of lysed cell or tissue samples using the KingFisher Cell and Tissue DNA Kit Cat No 97030196 and the KingFisher Duo with a 12 pin magnet head and Microtiter deep well 96 pl
28. 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 in several other enzymatic reactions The KingFisher Cell and Tissue DNA Kit is developed for the purification of DNA from cell cultures tissue samples and bacterial cultures using paramagnetic particles The purification process requires no phenol chloroform extraction or alcohol precipitation and needs very little hands on time The reagents and specific plastic consumables are designed to work with the KingFisher Flex KingFisher Duo or KingFisher mL magnetic particle processors as part of an integrated system The KingFisher Cell and Tissue DNA 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 Cell and Tissue DNA Kit for any particular use because the performance of the kits has not been validated for any specific organism The KingFisher Cell and Tissue DNA 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 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 11 Product Description Kit specifications magnetic particles Samples are lysed in the Lysis Buffer and
29. or KF magnet 100 pes 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 non sterile 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 Table B 3 Thermo Scientific KingFisher Duo consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 96 plate 50 pcs 97003510 KingFisher Duo 6 tip combs and KingFisher 24 deep well plate 48 pcs 12 pcs of 24 deep well plates each including 4 tips combs 97003520 KingFisher Duo elution strip 40 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 96 plate 1 box tips combs plates and elution strips for 96 samples Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Ordering Information Table B 4 Thermo Scientific KingFisher mL consumables Cat No Product Package size 97002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 20 x 45 pcs 97002131 KingFisher mL combi tubes and tip combs for 60 samples 60 97002141 KingFisher mL combi tubes and tip combs for 240 samples 240 Table B 5 Thermo Scientific KingFisher consumables Cat No Product Package size 97002070 KingFish
30. proximately for 3 minutes at 1200 rpm to pellet the cells to the bottom of the tube Remove the supernatant and add 200 ul of Lysis Buffer and 25 pl of Proteinase K working solution to each sample If the Proteinase K working solution has been added to the Lysis Buffer to form a premixture the solution should be used immediately Mix the samples thoroughly by pipetting up and down several times depending on the sample and vortex for 30 seconds or until the samples are lysed This is an essential step in the purification process and should be done carefully Briefly centrifuge the samples and incubate at 70 C for 10 15 minutes Occasional mixing during the incubation may enhance the lysis Follow the instructions below for clearing of the lysis mix Before clearing of the lysis an optional RNase A treatment can be done see below Lysing of tissue To lyse the tissue samples add 200 ul of Lysis Buffer and samples 25 ul of Proteinase K working solution to each sample If the Proteinase K working solution has been added to the Lysis Buffer to form a premixture the solution should be used immediately Vortex the samples for 15 seconds centrifuge briefly and incubate at 56 C until the samples are lysed Depending on the tissue sample size and structure lysing should take a minimum of one hour or even overnight Follow the instructions below for clearing of the lysis mix Before clearing of the lysis an optional RNase A treatment can be done
31. reatment are given in RNase A treatment on page 22 2 Place empty KingFisher mL tube strips on a tube strip tray Prepare the tubes that is starting from the first tube at the slip end of a tube strip Add the following reagents to the tubes Tube Tube name Content Reagent volume A Sample Lysed sample 225 ul KingFisher 25 ul Magnetic Beads Binding Buffer 360 pl B Wash 1 Wash Buffer 1 600 pl C Wash 2 Wash Buffer 2 600 pl D Wash 3 Wash Buffer 3 800 pl E Elution Elution Buffer 150 pl 30 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher mL for DNA purification from 225 ul of lysed cell or tissue samples 3 Prepare the KingFisher mL for the run Switch on the KingFisher mL and insert the tray into the instrument Insert the tip combs into their slots and close the front lid 4 Start the KF_TissueDNA_mL protocol with the KingFisher mL Connect the PC with BindIt Software 3 2 to the KingFisher mL Start the KF_TissueDNA_mL protocol When the KingFisher mL is to be run as a standalone instrument transfer the KF_TissueD NA_mL protocol to the KingFisher mL The instructions for transferring the protocol can be found in Chapter 4 Using the software in the BindIt Software for KingFisher Instruments version 3 2 User Manual 5 After the run is completed remove the
32. rolonged storage of the sample material may reduce the DNA yield If the KingFisher instrument mixes too strongly during the wash 3 it may cause partial elution of DNA already in the Wash Buffer 3 Use only Thermo Scientific KingFisher plates tubes or 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 Prolonged storage of the sample material may reduce the quality of the DNA Insufficient washing causes impurities in the Elution Buffer Wash Buffer 2 contains ethanol Carryover of the buffer may cause unsatisfactory performance in downstream applications Carryover of the KingFisher Magnetic Beads to the Elution Buffer may affect the A A ratio Make sure that the KingFisher Magnetic Beads do not affect the measurement by centrifuging the samples or placing 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 cause problems in most downstream processes Continued Thermo Fisher Scientific Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual 37 Troubleshooting Problem Magnetic particles remaining in the lysed sample or elution well Continued Possible cause and actions Starting material that is too viscose prevents efficient collection of the KingFisher Magnetic Beads from the lysed sample
33. should be 1 7 2 0 A Thermo Scientific NanoDrop can be used for the direct measurement of DNA without diluting the sample It is recommended that A329 correction be used for the absorbance values Subtract the A329 from the A260 and Ago ratios to remove the effects of carryover of the magnetic beads Use these calculations to measure the DNA content Concentration of DNA 50 pg ml x A260 A320 x dilution factor e Total amount of DNA concentration x volume of sample in ml e Purity of DNA A260 A320 A280 A320 32 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Chapter 6 General Information Reagent specificity A reagent must not be used with any kit other than that and volumes 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 KingFisher Duo User Manual or KingFisher mL User Manual to avoid spillover and to keep performance at the most efficient level Handl ing of The KingFisher Magnetic Beads have a tendency to magn etic beads sediment relatively quickly in the Binding Buffer Once a premixture of the beads and the Binding Buffer has been made the mixture should be used immediately 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
34. ticle processors Cat No Product 5400000 KingFisher magnetic particle processor 5400050 KingFisher mL magnetic particle processor 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 Discontinued KingFisher 96 magnetic particle processor Table 1 3 Thermo Scientific 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 non sterile 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 8 Thermo Scientific KingFisher Cell and Tissue DNA Kit Instruction Manual Thermo Fisher Scientific Table 1 4 Thermo Scientific KingFisher Duo consumables Kit Content Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 96 plate 50 pcs 97003510 KingFisher Duo 6 tip combs and KingFisher 24 deep well plate 48 pcs 12 pcs of 24 deep well plates each including 4 tips

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