KingFisher Total RNA Kit
Contents
1. Thermo Scientific KingFisher Total RNA Kit Instruction Manual Rev 1 2 Cat no N11999 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 PURPOSE THERM2. the Working Solutions ssssscscsssssssssssssssssssssssssessssseseesesaees 19 Storage conditions eiei a ei EE an 19 Preparation of the rDNase storage and working solutions 19 Preparation of the Reducing Agent TCEP working solution 20 Protocols and Pipetting Instructions 21 Handling of KingFisher Magnetic Beads 21 Homogenization of samples s sssssssssessssessssresressseresresresseseess 22 Homogenization of tissue samples cece eeeseeeeeeeeees 22 Homogenization of cell samples eseeseeeseeeeeeeeees 22 Instructions for KingFisher Flex with 96 deep well plates for total RNA purification from 350 ul of lysed cells or tissue 23 Summary of plate contents s ssssssssssssssessseessesssessresreesreesrees 26 Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for RNA purification of 350 pl lysed cell or tissue samples cecececeseeeseseseseseeeseseetetseeseeeeees 27 Summary of plate and elution strip contents eee 30 Instructions for KingFisher mL for total RNA purification from 350 ul of lysed cells or tise cesses eseseseseeeseeeeteeeees 31 Summary of tube contents Quantification and determination of the purity of RNA E 35 Thermo Scientific KingFisher Total RNA Kit Instruction Manual 5 Contents Chapter 6 Appendix A Appendix B General Information ssssssscssecssssssessessteestesseestesstesaeesseesseeass 37 Reagent spec
3. 7 Add the Binding Buffer to the DNase plate during the dispense step When the KingFisher Flex pauses at the dispense step after the DNase digestion step approximately 25 minutes after starting the run remove the DNase plate from the instrument and separately add 350 ul of Binding Buffer per well to the DNase plate to rebind the RNA Plate Plate Plate Content Reagent volume number type name per well 2 Microtiter DNase Binding Buffer 350 pl deep well 96 plate 8 Place the DNase plate back into the instrument and press Start After the pause the protocol will continue to the end 9 After the run is completed remove the plates and store the purified total RNA 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 total RNA accordingly The purified total RNA is ready for use in downstream applications The final RNA concentration in the Elution Buffer may increase if the purified RNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall RNA yield Thermo Scientific KingFisher Total RNA Kit Instruction Manual 25 Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for total RNA purification from 350 ul of lysed cells or tissue Summary of Table 5 1 Summary of plate contents plate contents Plate Plate Plate Content Sampl
4. remove the tube strip tray from the instrument and separately add 350 pl of Binding Buffer to each DNase tube to rebind the RNA Tube Tube name Content Reagent volume B DNase Binding Buffer 350 pl 7 Place the tube strip tray back into the instrument and press Start After the pause the protocol will continue to the end 8 After the run is completed remove the tube strips and store the purified total RNA When the protocol is completed remove the tubes and turn off the instrument Store the purified total RNA accordingly The purified total RNA is ready for use in downstream applications The final RNA concentration in the Elution Buffer may increase if the purified RNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall RNA yield Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 33 Protocols and Pipetting Instructions Instructions for KingFisher mL for total RNA purification from 350 ul of lysed cells or tissue Summary Of Table 5 3 Summary of tube contents tube contents Tube Tube name Content Sample reagent volume A Sample Homogenized 350 pl sample KingFisher 30 ul Magnetic Beads Binding Buffer 350 pl B DNase rDNase 300 pl working solution Dispense step add 350 ul of Binding Buffer to each of the DNase tubes B tubes during the pause in the Bindlt protocol C Wash1 Wash Buffer 1 600 ul D Wash2 Wa
5. 14 Thermo Scientific KingFisher Total RNA Kit Instruction Manual room temperature Thermo Fisher Scientific Product Description KingFisher magnetic particle processors Table 2 2 Overview of Thermo Scientific KingFisher systems KingFisher mL KingFisher Processing volume 50 1000 pl 20 200 ul Capacity Up to 15 samples per run Up to 24 samples per run max 2 x 10 cells or 20 mg of tissue per sample with the KingFisher Total RNA Kit Magnetic head 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 Thermo Fisher Scientific The BindIt Software 3 2 protocols optimized for the KingFisher Total RNA Kit are available for the KingFisher Flex the KingFisher Duo and the KingFisher mL 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 Scientific KingFisher Total RNA Kit Instruction Manual 15 Product Description KingFisher magnetic particle processors 16 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Chapter 3 Safety Information The following components of the KingFisher Total RNA
6. magnetic beads Binding and wash steps Thermo Fisher Scientific Chapter 6 General Information 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 KingFisher Duo User Manual or KingFisher mL User Manual to avoid spillover and to keep performance at the most efficient level The KingFisher Magnetic Beads have a tendency to sediment relatively quickly in the Binding Buffer Once a premixture of the beads and 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 yield of the purified RNA A detergent reagent Tween 20 is used in the wash 3 step Avoid vigorous shaking of the bottle as this will cause foam to form on the surface of the reagent leading to problems while transferring the correct amount of the buffer to the respective wells or tubes The binding between the RNA and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt but chaotropic salts are not present in the wash 3 step and accordingly the binding is weak Avoid strong mixing speeds and releasing the beads into the wash 3 step in order to minimize the loss of RNA during the wash step A short wash and
7. 96 samples in the KingFisher Flex or 12 samples in the KingFisher Duo and for the purification of 15 samples in the KingFisher mL The obtained total RNA can be used directly in various downstream applications Up to 2 x 106 cells or 20 mg of tissue per sample can be used as sample material The yields of acquired purified RNA depend on the sample type the method of sample storage and the method of tissue disruption The KingFisher Total RNA Kit can be processed completely at room temperature 12 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific KingFisher magnetic particle processors Thermo Fisher Scientific Product Description KingFisher magnetic particle processors The KingFisher Magnetic Beads are highly reactive superparamagnetic beads The binding capacity is approximately 1 ug of total RNA per 1 pl of KingFisher Magnetic 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 or tubes Use only Thermo Scientific KingFisher plastic consumables Use of products from other manufacturers may cause unsuitable mixing or even instability in the KingFisher instrument The instrument functions without any dispensing or a
8. 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 Lysis Buffer Hazardous contents Guanidine thiocyanate lt 60 Safety instructions Harmful by inhalation in contact with skin and if swallowed Contact with acids liberates very toxic gas Harmful for aquatic organisms may cause long term adverse effects in the aquatic environment Keep away from food drink and animal feed Binding Buffer lsopropanol gt 90 Highly flammable Irritating to eyes Vapors may cause drowsiness and dizziness Keep container tightly closed Keep away from sources of ignition no smoking Avoid contact with skin and eyes In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wash Buffer 1 Guanidine thiocyanate lt 30 and ethanol lt 45 Flammable Harmful by inhalation and in contact with skin Keep away from food drink and animal feed Keep away from sources of ignition no smoking Wash Buffer 2 Ethanol lt 90 Highly flammable Keep container tightly closed Keep away from sources of ignition no smoking rDNase Lyophilized rDNase May cause sensitization by inhalation and skin contact Do not inhale
9. 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 38 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Thermo Fisher Scientific General Information Elution step To gain a maximal yield of purified total RNA 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 RNA from the beads 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 Thermo Scientific KingFisher Total RNA Kit Instruction Manual 39 General Information Elution step 40 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Table A 1 Troubleshooting guide Problem Low total RNA yield Appendix A Troubleshooting Possible cause and actions There s
10. increase if Thermo Scientific KingFisher Total RNA Kit Instruction Manual 29 Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for RNA purification of 350 ul lysed cell or tissue samples the purified RNA is eluted into a smaller volume of the buffer than is recommended but this can slightly reduce the overall RNA yield Summary of plate and Table 5 2 Summary of plate and elution strip contents elution strip contents Plate name and type Row Row name Content Reagent Sample volume per well Total RNA plate A Sample Homogenized 350 pl sample Microtiter deep well 96 plate KingFisher 30 pl Magnetic Beads Binding Buffer 350 pl Tip 12 tip comb Empty Empty Empty Empty DNase rDNase working 300 pl solution Dispense step add 350 ul of Binding Buffer per well to row D during the pause in the Bindlt protocol E Wash 1 Wash Buffer 1 600 ul F Wash 2_1 Wash Buffer 2 900 pl G Wash 2_2 Wash Buffer 2 900 ul H Wash 3 0 02 Tween 20in 1000 ul RNase free water Elution strip Elution Elution Buffer 100 pl 30 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher mL for total RNA purification from 350 ul of lysed cells or tissue Instructions for These instructions are for the total RNA purification from KingFisher mL 350 pl of lysed cells or tis
11. 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 Up to 24 samples Up to 12 samples Up to 6 samples per run max 2 x per run per run per run 10 cells or 20 mg of tissue per sample with the KingFisher Total RNA Kit Magnetic head 96 interchangeable formats for PCR plate KingFisher Flex 96 KF plate Microtiter deep well 96 plate 24 format for KingFisher Flex 24 deep well plate 12 pin magnet head for Microtiter deep well 96 plate 6 pin magnet head for KingFisher Flex 24 deep well plate Plates KingFisher Flex 96 KingFisher Flex Microtiter deep well KingFisher Flex 24 KF plate 20 200 pl 24 deep well plate 96 plate 50 1000 pl deep well plate 96 well PCR plate 200 5000 yl KingFisher Duo 200 5000 pl skirted 20 100 ul elution strip Microtiter deep well 30 130 ul 96 plate 50 1000 ul Tip combs KingFisher Flex 96 KingFisher Flex KingFisher Duo KingFisher Duo tip comb for PCR 24 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 C to 75 C in to 115 C
12. 2 900 pu deep wel 96 plate 5 Microtiter Wash2_2 Wash Buffer 2 900 p deep wel 96 plate 6 Microtiter Wash3 0 02 Tween20 1000 pl deep well in RNase free 96 plate water 7 KingFisher Elution Elution Buffer 150 pl Flex 96 KF plate 5 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate that is an empty KingFisher Flex 96 KF plate 6 Start the KF_TotRNA_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_TotRNA_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 24 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for total RNA purification from 350 ul of lysed cells or tissue Thermo Fisher Scientific When the KingFisher Flex is to be run as a standalone instrument transfer the KF_TotRNA_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
13. 97020196 KingFisher Total RNA 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 for 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 Total RNA 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 KingF
14. KingFisher Total RNA Kit is of high quality and free of proteins nucleases and other contaminants or inhibitors Purified total RNA is suitable for direct use in many different downstream applications such as PCR polymerase chain reaction after reverse transcription and in several other enzymatic reactions The KingFisher Total RNA Kit is developed for the purification of total RNA from cell and tissue samples 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 Total RNA 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 Total RNA Kit for any particular use because the performance of the kits has not been validated for any specific organism The KingFisher Total RNA Kit uses magnetic particle technology for total RNA purification The Thermo Scientific KingFisher technology combines the speed and efficiency of RNA purification with easy handling of magnetic particles It is recommended that tissue Thermo Scientific KingFisher Total RNA Kit Instruction Manual 11 Product Description Kit specifications Ki
15. O 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 sssssssssssssssssessessessessessessessssseesseseseesaeees 17 Storage Conditions and Preparation of
16. Thermo Scientific KingFisher magnetic particle 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 Total RNA 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 wel
17. a slow mixing speed without releasing the beads into the buffer are recommended Thermo Scientific KingFisher Total RNA Kit Instruction Manual 37 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 RNA The volume of the Elution Buffer can be modified depending on the user requirements concerning the purified RNA concentration The final RNA concentration in the Elution Buffer may increase if the purified RNA is eluted into a smaller volume of the buffer but this can slightly reduce the overall RNA yield The modifications of the volumes in the elution step must be done in BindIt 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 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
18. dust Avoid contact with skin Reducing Agent TCEP Thermo Fisher Scientific TCEP Tris 2 carboxyethyl phosphine hydrochloride Irritating to eyes and 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 KingFisher Total RNA Kit Instruction Manual 17 Safety Information 18 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Chapter 4 Storage Conditions and Preparation of the Working Solutions Storage conditions All buffers and reagents included in the KingFisher Total RNA 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 Preparation of To prepare the rDNase storage solution for the KingFisher the rDNase sto rage Total RNA Kit add 800 pl of RNase free water to each ki vial of the lyophilized rDNase and incubate at room and i ing temperature for 1 minute Occasional gentle rotation of solutions the vial enhances dissolvement of the rDNase but avoid forceful mixing The rDNase storage solution should be stored at 20 C where it remains stable for 6 months Do not freeze and thaw the rDNase storage solution more than three times Calculate the amount of rDNase working solution needed For the purification of one sample mix 25 pl of rDNase storage solution and 275 ul of rDNase Buffe
19. e reagent number type name volume per well 1 Microtiter Sample Homogenized 350 u deep well sample 96 plate KingFisher 30 u Magnetic Beads Binding Buffer 350 p 2 Microtiter DNase rDNase 300 p deep well working solution 96 plate Dispense step add 350 ul of Binding Buffer per well to the DNase plate Plate 2 during the pause in the Bindlt protocol 3 Microtiter Wash1 Wash Buffer 1 600 ul deep well 96 plate 4 Microtiter Wash 2_1 Wash Buffer 2 900 pl deep well 96 plate 5 Microtiter Wash 2_2 Wash Buffer 2 900 ul deep well 96 plate 6 Microtiter Wash3 0 02 Tween 20 1000 pl deep well in RNase free 96 plate water 7 KingFisher Elution Elution Buffer 150 pl Flex 96 KF plate 8 KingFisher Tip Plate Flex 96 KF plate 26 Thermo Scientific KingFisher Total RNA 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 RNA purification of 350 ul lysed cell or tissue samples Instructions for These instructions are for the RNA purification from KingFisher Duo 350 pl of lysed cell or tissue samples using the KingFisher ith 12 pi t Total RNA Kit Cat No 97020196 and the KingFisher wi pin magne Duo with a 12 pin magnet head and Microtiter deep well head and 96 deep 96 plates well plates for ae 2 When using the KingFisher Total RNA Kit for the first RNA purification time prepare the stora
20. etting Instructions Instructions for KingFisher mL for total RNA purification from 350 ul of lysed cells or tissue Tube Tube name Content Sample reagent volume A Sample Homogenized 350 pl sample KingFisher 30 pl Magnetic Beads Binding Buffer 350 pl B DNase rDNase 300 pl working solution Wash1 Wash Buffer 1 600 pl Wash2 Wash Buffer 2 900 ul E Elution Elution Buffer 150 pl 4 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 5 Start the KF_TotRNA_mL protocol with the KingFisher mL Connect the PC with BindIt Software 3 2 to the KingFisher mL Start the KF_TotRNA_mL protocol When the KingFisher mL is to be run as a standalone instrument transfer the KF_TotRNA_mL protocol to the KingFisher mL The instructions for transferring the protocol can be found in Chapter 4 Using the software in the BindlIt Software for KingFisher Instruments version 3 2 User Manual 32 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher mL for total RNA purification from 350 ul of lysed cells or tissue 6 Add the Binding Buffer to the DNase tubes during the dispense step When the KingFisher mL pauses at the dispense step after the DNase digestion step approximately 25 minutes after starting the run
21. ge and working solutions for rDNase of 350 pl lysed cell and Reducing Agent TCEP For more instructions refer or tissue samples to Chapter 4 Storage Conditions and Preparation of the Working Solutions 1 Homogenize the samples according to the instructions given in Homogenization of samples on page 22 2 Prepare the rDNase working solution for the samples that are used in a run The working solution should be used immediately First calculate the amount of rDNase working solution needed For the purification of one sample mix 25 ul of rDNase storage solution and 275 pl of rDNase Buffer solution 3 Take one empty Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip 4 Prepare the Total RNA 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 row C is left empty Resuspend the KingFisher Magnetic Beads well e g by vortexing before removing them from the bottle Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 27 Protocols and Pipetting Instructions Instructions for KingFisher Duo with 12 pin magnet head and 96 deep well plates for RNA purification of 350 ul lysed cell or tissue samples Plate name and type Row Row name Content Reagent Sample volume per well Total RNA plate A Sample Homogenized 350 pl sample Microtiter deep we
22. hould 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 Efficient lysis of the cells or tissue increases the total RNA yield Prolonged storage of the sample material may reduce the total RNA yield If the mixing is too strong it may cause partial elution of total RNA already during the wash 3 step Use only Thermo Scientific KingFisher plates or tubes with the KingFisher instruments Use of products from other manufacturers may cause unsuitable mixing and affect the yield of purified total RNA Low purity Prolonged storage of the sample material may reduce the quality and quantity of the total RNA Insufficient washing causes impurities in the Elution Buffer The Wash Buffers 1 and 2 contain 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 affect most downstream processes Continued Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instructi
23. ificity and volumes cece eeeeeeeeeeeeeeeeeeeeeeees 37 Handling of magnetic beads oo eeeeeeeeeeeeeeeeeeeeeeeeees 37 Binding and wash steps ccceeeesesesesseeeeeeeeeeeeseeseeeseseseeeeees 37 Elution Stepan a Qian ie ae 38 Troubleshooting sssssscsesssssssssssessessesessssesssssessesseaesseaeeseeseaees m Ordering Information ssssssssssssssssssssesssssssassssssessseseesseees 43 6 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Chapter 1 Kit Content Table 1 1 Thermo Scientific KingFisher Total RNA Kit Item KingFisher Total RNA Kit 1x 96 Cat No 97020196 Package size 1x 96 samples KingFisher Magnetic Beads 3 1 ml rDNase 3 vials rDNase Buffer 35 ml Reducing Agent TCEP 1 vial Lysis Buffer 40 ml Binding Buffer 75 ml Wash Buffer 1 65 ml Wash Buffer 2 200 ml Elution Buffer 20 ml RNase free water 120 ml The KingFisher Total RNA Kit Cat No 97020196 is intended for the purification of cell or 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 Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 7 Kit Content Equipment and reagents to be supplied by the user e Tween 20 e Magnetic particle processor e Homogenization equipment optional Table 1 2
24. isher 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 KingFisher 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 44 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Notes Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 45 Notes 46 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Thermo Fisher Scientific Oy Ratastie 2 P O Box 100 Fl 01621 Vantaa Finland www thermoscientific com N11999
25. l 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 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 Total RNA Kit Instruction Manual 9 Kit Content 10 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Introduction Intended use Principle and procedure Thermo Fisher Scientific Chapter 2 Product Description The KingFisher Total RNA Kit is designed for rapid automated purification of total RNA from cell and tissue samples using KingFisher instruments The total RNA can be purified from up to 2 x 106 cells or 20 mg of tissue The total RNA purified with the
26. ll 96 plate KingFisher 30 pl Magnetic Beads Binding Buffer 350 pl B Tip 12 tip comb Empty C Empty Empty Empty D DNase rDNase working 300 pl solution E Wash 1 Wash Buffer 1 600 pl F Wash 2_1 Wash Buffer 2 900 pl G Wash 2_2 Wash Buffer 2 900 ul H Wash 3 0 02 Tween 20in 1000 pl RNase free water 5 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 6 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on a Total RNA plate 7 Start the KF_totRNA_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_totRNA_Duo protocol Insert the Total RNA plate and elution strip into 28 Thermo Scientific KingFisher Total RNA 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 RNA purification of 350 ul lysed cell or tissue samples Plate name and type Total RNA plate Micr
27. on Manual 41 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 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 41 KingFisher Magnetic Beads that occasionally remain attached to the tip combs at the end of the process do not affect the total RNA yield as the RNA 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 42 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Appendix B Ordering Information Table B 1 Thermo Scientific KingFisher Total RNA Kits Cat No Product Package size
28. otiter deep well 96 plate Thermo Fisher Scientific 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 the KF_totRNA_Duo protocol to the KingFisher Duo The instructions for transferring the protocol can be found in the Bindlt Software for KingFisher Instruments version 3 2 User Manual 8 Add the Binding Buffer to row D during the dispense step When the KingFisher Duo pauses at the dispense step after the DNase digestion step approximately 25 minutes after starting the run remove the DNase plate from the instrument and separately add 350 ul of Binding Buffer per well to row D to rebind the RNA Row Row name Content Reagent Sample volume per well D DNase Binding Buffer 350 pl 9 Place the Total RNA plate back into the instrument and press OK After the pause the protocol will continue to the end 10 After the run is completed remove the plate and store the purified RNA 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 RNA accordingly The purified RNA is ready for use in downstream applications The final RNA concentration in the Elution Buffer may
29. r solution The rDNase working solution should be used immediately after preparation Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 19 Storage Conditions and Preparation of the Working Solutions Preparation of the Reducing Agent TCEP working solution Preparation of To prepare the Reducing Agent TCEP working solution the Reduci ng ae the a ae i Kit add 750 ul of ee ree water to a vial of Reducing Agent TCEP Incubate Agent TCEP the vial at room temperature re sae minutes and wo rking solution mix occasionally to dissolve the Reducing Agent TCEP completely The Reducing Agent TCEP working solution should be stored at 20 C 20 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Chapter 5 Protocols and Pipetting Instructions Before beginning the total RNA purification protocol carefully read through the Thermo Scientific KingFisher Flex User Manual Cat No N07669 the Thermo Scientific KingFisher Duo User Manual Cat No N12420 or the Thermo Scientific KingFisher mL User Manual Cat No 1508260 and the Thermo Scientific BindIt 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 Handl ing of a homogeneous distribution of the KingFisher Magnetic Kin gFisher Beads in the container is essential before the beads are Magnetic Bead
30. s 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 Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 21 Protocols and Pipetting Instructions Homogenization of samples Homogenization Use up to 2 x 10 of cultured cells or 20 mg of tissue per of samples sample Efficient homogenization of the sample is an essential step before RNA purification in order to gain a good yield of high quality RNA Tissue sample can be homogenized with various kinds of homogenizers such as bead mills The homogenization step must disrupt the structures of the sample material completely in order to ensure a high yield of RNA Homogenization of To homogenize tissue samples add 350 ul of Lysis Buffer tissue samples and 6 pil of Reducing Agent TCEP working solution to each sample and use a suitable homogenization instrument to disrupt the tissue After the tissue has been homogenized centrifuge the sample briefly 30 seconds 1500 x g and transfer the supernatant to a Thermo Scien
31. sh Buffer 2 900 ul E Elution Elution Buffer 150 pl 34 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Quantification and determination of the purity of RNA Thermo Fisher Scientific Protocols and Pipetting Instructions Quantification and determination of the purity of RNA It is recommended to measure the absorbance at 320 nm 280 nm and 260 nm The concentration of RNA can be defined with the absorbance at 260 nm A260 One unit at 260 nm corresponds to 40 pg of RNA per ml The ratio between the A260 A280 indicates the purity of the RNA and the value for RNA should be gt 1 8 2 1 A Thermo Scientific NanoDrop can be used for the direct measurement of RNA without diluting the sample It is recommended that A320 correction is used for the absorbance values Subtract the A329 from the A260 and Aggo ratios to remove the effects of carryover of magnetic beads Use these calculations to measure the RNA content Concentration of RNA 40 ug ml X A260 A320 x dilution factor Total amount of RNA concentration x volume of sample in ml e Purity of RNA A260 A320 A230 A320 Thermo Scientific KingFisher Total RNA Kit Instruction Manual 35 Protocols and Pipetting Instructions Quantification and determination of the purity of RNA 36 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Reagent specificity and volumes Handling of
32. spiration 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 and 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 total RNA 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 The KingFisher Total RNA Kit is optimized and ready for use with KingFisher systems Thermo Scientific KingFisher Total RNA Kit Instruction Manual 13 Product Description KingFisher magnetic particle processors The KingFisher magnetic particle processors are intended for professional research use by trained personnel Detailed information and user instructions for the KingFisher instruments can be found in their respective user
33. structions refer to Chapter 4 Storage Conditions and Preparation of the Working Solutions 1 Homogenize the samples according to the instructions given in Homogenization of samples on page 22 2 Prepare the rDNase working solution for the samples that are used in a run and should be used immediately First calculate the amount of rDNase working solution needed For the purification of one sample mix 25 pl of rDNase storage solution and 275 pl of rDNase Buffer solution 3 Take six empty Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates 4 Prepare and fill six 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 Total RNA Kit Instruction Manual 23 Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for total RNA purification from 350 ul of lysed cells or tissue Plate Plate Plate Content Sample reagent number type name volume per well 1 Microtiter Sample Homogenized 350 ul deep well sample 96 plate KingFisher 30 pl Magnetic Beads Binding Buffer 350 u 2 Microtiter DNase rDNase 300 p deep wel working solution 96 plate 3 Microtiter Wash1 Wash Buffer 1 600 p deep wel 96 plate 4 Microtiter Wash2_1 Wash Buffer
34. sue using the KingFisher Total for total RNA RNA Kit Cat No 97020196 and the KingFisher mL purification from When using the KingFisher Total RNA Kit for the first 350 pl of lysed time prepare the storage and working solutions for rDNase cells or tissue and Reducing Agent TCEP For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Working Solutions A tube strip tray in the KingFisher mL may contain up to 15 separate Thermo Scientific KingFisher 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 Homogenize the samples according to the instructions given in Homogenization of samples on page 22 2 Prepare the rDNase working solution for the samples that are used in a run and should be used immediately First calculate the amount of rD Nase working solution needed For the purification of one sample mix 25 yl of rDNase storage solution and 275 pl of rDNase Buffer solution 3 Place empty KingFisher mL tubes 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 Thermo Fisher Scientific Thermo Scientific KingFisher Total RNA Kit Instruction Manual 31 Protocols and Pip
35. t specifications samples are mechanically disrupted in the Lysis Buffer before the purification can start to ensure a good yield of purified RNA In case of cells the sample should be pelleted before the addition of the Lysis Buffer The samples are sedimented after a short centrifugation step and the cleared lysates are transferred to Thermo Scientific KingFisher plates for processing with a Thermo Scientific KingFisher magnetic particle processor RNA binds to the surface of the Thermo Scientific KingFisher Magnetic Beads in the presence of a chaotropic salt Co purified DNA is removed during DNase treatment The following effective wash steps dispose of proteins cell debris and any residual contaminants while the RNA bound to the KingFisher Magnetic Beads is transferred through the wash steps Two different Wash Buffers are used followed by a rapid rinse in 0 02 Tween 20 in RNase free water or an air drying step which considerably improves the purity of the total RNA High quality total RNA is eluted into the Elution Buffer and is ready for subsequent downstream processes such as enzymatic reactions The KingFisher Total RNA Kit is designed for rapid automated preparation of highly pure total RNA from cell and tissue samples using Thermo Scientific KingFisher magnetic particle processors When excluding a dispense step requiring the addition of the Binding Buffer the approximate processing time is 60 minutes for the purification of
36. tific Microtiter deep well 96 plate or a Thermo Scientific KingFisher mL tube and begin the purification of RNA using the KingFisher Flex the KingFisher Duo or the KingFisher mL See the detailed instructions below Homogenization To homogenize cell samples add 350 ul of Lysis Buffer of cell samples and 6 ul of Reducing Agent TCEP working solution to a pelleted cell sample Use a syringe or pipette to break the pellet by aspirating several times up and down and centrifuge briefly 30 seconds 1500 x g Transfer the supernatant to a Microtiter deep well 96 plate or a KingFisher mL tube and begin the purification of RNA using the KingFisher Flex the KingFisher Duo or the KingFisher mL See the detailed instructions below 22 Thermo Scientific KingFisher Total RNA Kit Instruction Manual Thermo Fisher Scientific Protocols and Pipetting Instructions Instructions for KingFisher Flex with 96 deep well plates for total RNA purification from 350 ul of lysed cells or tissue Instructions for These instructions are for the total RNA purification from KingFisher Flex 350 pl of lysed cells or tissue using the KingFisher Total RNA Kit Cat No 97020196 and the KingFisher Flex with 96 deep well with Microtiter deep well 96 plates plates for total RNA purification When using the KingFisher Total RNA Kit for the first time prepare the storage and working solutions from 350 ul of lysed for rDNase and Reducing Agent TCEP For more cells ortissue in
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