KingFisher Pure RNA Plant Kit Instruction Manual
Contents
1. Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 0 20 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 1 Take one empty Microtiter deep well 96 plate and one Thermo Scientific TM KingFisher Duo elution strip 2 Prepare the Plant RNA plate i e a Microtiter deep well 96 plate Add the following reagents to the rows Note that row B is reserved for the tip comb and should be left empty Note that row C is also left empty Sample name and Row Row name Content reagent volume per well PlantRNA A DNase DNase working 200 pl plate solution Microtiter B Tip 12 tip comb Empty deep well c Empty Empty Empty 96 plate D Wash 2_2 Wash Buffer 2 700 ul E Wash 2_1 Wash Buffer 2 700 ul F Wash 1_2 Wash Buffer 1 700 ul G Wash 1_1 Wash Buffer 1 900 ul H Sample Plant lysate 400 pl KingFisher 25 pul Magnetic Beads Ethanol 400 pl Resuspend the KingFisher Magnetic Beads well by vortexing before use 3 Fill the KingFisher Duo elution strip as follows Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user and the nuclease free water is pipetted into the correct wells2. volume approx volume 1 ml 200 ul 1 ml 200 ul Magnetic head 96 inter 24 format for 12 pin magnet 6 pin magnet changeable KingFisher Flex head for head for formats for 24 deep well Microtiter deep KingFisher Flex Microtiter deep plate well 96 plate 24 deep well well 96 plate plate PCR plate and KingFisher Flex 96 KF plate Plates KingFisher Flex KingFisher Flex Microtiter deep KingFisher Flex 96 KF plate 24 deep well well 96 plate 24 deep well 20 200 ul plate 50 1000 ul plate 96 well PCR 200 5000 ul KingFisher Duo 200 5000 pul plate skirted elution strip 20 100 ul 80 130 ul Microtiter deep well 96 plate 50 1000 pul Tip combs KingFisher Flex KingFisher Flex KingFisher Duo KingFisher Duo 96 tip comb for 24 tip comb for 12 tip comb 6 tip comb PCR magnets deep well KingFisher Flex magnets tip comb for KF magnets KingFisher Flex 96 tip comb for deep well magnets Heating Heating block temperature from Heating block temperature from temperature 5 C above ambient room 10 C to 75 C elution strip temperature to 115 C 4 C to 75 C at room temperature See the details above on the Plates row Thermo Fisher Scientific Safety Information The following components of the KingFisher Pure RNA Plant Kit contain hazardous contents Table 3 1 Always wear a laboratory coat disposable gloves and goggles and follow the safety instructions provided in the kit instruc
3. Revision 1 0 N14039 March 2013 Copyright 2013 Thermo Fisher Scientific Inc All rights reserved Tween is a trademark of ICI Americas Inc Virkon is a trademark of E l du Pont de Nemours and Company or its affiliates All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Reproduction of the accompanying user documentation in whole or in part is prohibited Disclaimer Thermo Fisher Scientific reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without prior notice as part of continuous product development Although this manual has been prepared with every precaution to ensure accuracy Thermo Fisher Scientific assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information This instruction manual supersedes all previous editions Products are for Research Use Only Not for use in diagnostic procedures The Product will operate substantially in conformance with Thermo Fisher Scientific s published specifications THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES WHETHER EXPRESSED OR IMPLIED ORAL OR WRITTEN WITH RESPECT TO THE PRODUCTS INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF PRODUCT QUALITY CONDITION DESCRIPTION MERCHANTABILITY OR FITNESS FOR ANY PARTICULAR PURPOSE THERMO FISHER SCIENTIFIC DOES NOT WARRANT THA
4. The KingFisher instrument family comprises four systems covering working volumes from 20 to 5000 ul Each system consists of an instrument specially designed plastic consumables and the easy to use Bindlt Software 3 2 The KingFisher Pure RNA Plant Kit is optimized and ready for use with the KingFisher Flex or KingFisher Duo 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 manuals The Bindlt Software 3 2 protocols optimized for the KingFisher Pure RNA Plant Kit are available for the KingFisher Flex and KingFisher Duo For more information go to www thermoscientific com kingfisherinfo or contact your local authorized distributor Thermo Fisher Scientific Thermo Scientific KingFisher Pure RNA Plant Kit 11 Chapter 2 Product Description 12 Thermo Scientific KingFisher Pure RNA Plant Kit Table 2 1 Overview of KingFisher Flex and KingFisher Duo magnetic particle processors KingFisher Flex KingFisher Duo 96 formats 24 format 12 format 6 format Processing volume 20 1000 pul 200 5000 ul 30 1000 ul 200 5000 pul Capacity Up to 96 Up to 24 Up to 12 Up to 6 samples per samples per samples per samples per run sample run sample run sample run sample volume approx volume approx
5. A A indicates the purity of the RNA The value for 260 280 RNA should be gt 1 8 2 1 It is recommended that A correction is used for the absorbance values Subtract the A from the A and A ratios to remove the effects of carryover of the magnetic particles e Concentration of RNA sample 40 ug ml x A Agog X dilution factor 260 e Total amount of RNA isolated concentration x volume of sample in ml e Purity of RNA sample A Aj Asan Ar Thermo Scientific KingFisher Pure RNA Plant Kit 23 Thermo Fisher Scientific General Information Reagent Specificity and Volumes A reagent must not be used with any kit other than that for which it is intended lt is strongly recommended that the volume of reagents in each well or tube is kept within the limits specified in the KingFisher Flex User Manual or KingFisher Duo User Manualto avoid spillover and to maximize efficiency of performance Handling of Magnetic Beads The KingFisher Magnetic Beads should be mixed thoroughly before use to avoid the risk of transferring variable amounts of the beads to the respective wells or tubes The amount of beads in the wells or tubes affects the yield of the purified total RNA Binding Wash and Elution Steps The binding between the purified RNA and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt The binding will remain throughout the wash steps until the elution where t
6. particular use as the performance of the kits has not been validated for any specific organism or downstream application Thermo Scientific KingFisher Pure RNA Plant Kit 9 Chapter2 Product Description Principle and Procedure The KingFisher Pure RNA Plant 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 The purification process requires no phenol chloroform extraction and needs very little hands on time To ensure a good yield of purified RNA the plant samples should be mechanically disrupted efficiently before beginning the purification The homogenized samples are resuspended into the Lysis Buffer Then he samples are incubated and sedimented using a short centrifugation The cleared lysates are transferred to the Thermo Scientific KingFisher plates for processing with a KingFisher magnetic particle processor The first step of the protocol further lyses the samples after which the RNA can bind to the surface of the KingFisher Magnetic Beads in the presence of the Binding Buffer The KingFisher Magnetic Beads are highly reactive superparamagnetic beads Copurified 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 transfer
7. 0 C Store the Thermo Scientific KingFisher Magnetic Beads at 4 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to two years from the manufacturing date Additional Reagents Required e 96 100 ethanol EtOH molecular biology grade e 2MDTT dithiothreitol solution e Polyvinylpyrrolidone PVP for woody lignified or polyphenol rich samples Table 1 2 Thermo Scientific KingFisher magnetic particle processors Cat No Product 5400100 KingFisher Duo magnetic particle processor 5400630 KingFisher Flex magnetic particle processor with 96 deep well head Table 1 3 Thermo Scientific KingFisher Flex consumables Cat No Product Package size 97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs 97002540 KingFisher Flex 96 KF plate 200 ul 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 6 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Kit Content Chapter 1 Table 1 4 Thermo Scientific KingFisher Duo consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003520 KingFisher Duo elution strip 40 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Mic
8. 7669 or the Thermo Scientific KingFisher Duo User Manual Cat No N12420 and the 7hermo Scientific Binalt Software for KingFisher Instruments version 3 2 User Manual Cat No NO7974 Bindlt Software protocols for the KingFisher Pure RNA Plant Kit can be found in Bindlt Software 3 2 and at www thermoscientific com kingfisher Handling of KingFisher Magnetic Beads A homogeneous distribution of the KingFisher Magnetic Beads in the container is essential before the beads are transferred to the wells in order to ensure a high consistency between the wells To gain complete resuspension of the beads shake the container vigorously or vortex briefly Avoiding Ribonuclease RNase Contamination RNA purity and integrity is essential for downstream applications RNase is a ubiquitously found enzyme which degrades RNA RNases are highly stable contaminants found in any laboratory environment Keep all kit components tightly sealed when not in use Skin is a common source of RNases Always wear gloves when handling reagents and RNA samples Use sterile RNase free pipette tips when working with RNA Remove RNase contamination from work surfaces and non disposable items pipettes centrifuges with reagents designed to remove RNase Thermo Scientific KingFisher Pure RNA Plant Kit 17 Chapter5 Protocols and Pipetting Instructions Homogenization of Sample Material Efficient homogenization of the sample material is an essen
9. 96 100 ethanol to each bottle 96 samples Cat No 98060196 and 384 samples Cat No 98060496 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 125 ml 50 ml Ethanol 96 100 125 ml 200 ml Total volume 250 ml 250 ml After preparing each solution mark the bottle to indicate that the step has been completed The buffers can be stored at room temperature Preparation of the DNase I Storage Solution To prepare the DNase storage solution add 440 ul of DNase Reconstitution Buffer to each vial of the lyophilized DNase Incubate at room temperature for 5 min Occasional gentle rotation of the vial helps to dissolve the DNase but avoid forceful mixing Store the DNase storage solution at 20 C Repeated freezing and thawing should be avoided Preparation of the DNase Working Solution Before each RNA purification calculate the amount of DNase working solution needed For the purification of one sample mix 100 ul of 2 x DNase Buffer 20 ul of Manganese Chloride Solution 4 ul of DNase storage solution and 76 ul of nuclease free water The DNase working solution should be used immediately after preparation 16 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Before beginning the total RNA purification protocol carefully read through the Thermo Scientific KingFisher Flex User Manual Cat No NO
10. Description Chapter 2 KingFisher Magnetic Particle Processors The KingFisher magnetic particle processors are designed for the automated transfer and processing of magnetic particles in microplate format The patented technology of the Thermo ScientificTM KingFisherTM systems is based on the use of magnetic rods covered with a disposable specially designed tip comb and plates or tubes Use only Thermo ScientificTM KingFisher plastic consumables as use of products from other manufacturers may cause unsuitable mixing or even instability in the KingFisher instrument The instrument functions without any dispensing or aspiration parts or devices Samples and reagents including magnetic particles are dispensed onto the plates according to the corresponding instructions Dispensing can be carried out manually or partially automatically using automatic dispensers for example the Thermo Scientific Multidrop Combi and or the Thermo Scientific Versette Thermo Scientific Bindlt Software 3 2 can be used for running ready made and optimized protocols for the Thermo Scientific KingFisher Pure Kits It is also possible to transfer the developed protocol onto the onboard software and run it directly from the instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting in high purity total RNA without risk of carryover or cross contamination
11. Fisher Flex 96 tip comb for deep well magnets on a Tip Plate i e an empty KingFisher Flex 96 KF plate 4 Start the PURE_RNAPlant_Flex96 protocol using the KingFisher Flex 96 and load the plates as instructed on the KingFisher Flex 96 instrument display Switch on the KingFisher Flex making sure that you are using the Thermo Scientific KingFisher Flex 96 deep well head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Flex Start the PURE_RNAPlant_Flex96 protocol Insert the Tip Plate and the filled plates into the instrument as indicated on the KingFisher Flex display After all the plates have been loaded into the instrument the protocol will start When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_RNAPlant_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Sindit Software for KingFisher Instruments version 3 2 User Manual 5 Add Rebinding Buffer and ethanol to the Sample plate during the dispense step Thermo Scientific KingFisher Pure RNA Plant Kit 19 Chapter5 Protocols and Pipetting Instructions When the KingFisher Flex pauses at the dispense step after the lysis step at approximately 25 minutes after starting the protocol run remove the DNase plate from the instrument and add the Rebinding Buffer and ethanol to the DNase plate to rebind the RNA One can make
12. G 216 conca rn a 15 Preparation of the Lysis Buffer 0oooooommm m o o o 15 Preparation of the Wash Buffers ooooomoo o o o o 15 Preparation of the DNase Storage SolutioN 16 Preparation of the DNase Working Solution 16 Protocols and Pipetting Instructions 55 17 Handling of KingFisher Magnetic BeadS 17 Avoiding Ribonuclease RNase Contamination 17 Homogenization of Sample Material 18 Instructions for KingFisher Flex with 96 Deep Well Plates 18 Instructions for KingFisher Duo with 12 pin Magnet Head 20 Quantification and Determination of the Purity of RNA 23 General Information 220000ceeeeee eee eens 25 Reagent Specificity and VoluMES o o oooommm 25 Handling of Magnetic Beads 0 eee cece eee 25 Binding Wash and Elution Steps 0 00 cee eee ee 25 Troubleshooting 0 0cee cece cece eee e nn nennen 27 Ordering Information 00ccc nennen eee 29 NOTE For more details on storing the kit reagents refer to Storage Conditions on page 6 Thermo Scientific KingFisher Pure RNA Plant Kit 3 Thermo Fisher Scientific Kit Content Table 1 1 Thermo Scientific KingFisher Pure RNA Plant Kit Item KingFisher Pure RNA Plant Kit Cat No 98060196 98060496 Package siz
13. Reagent volume per well Elution strip Content KingFisher Duo elution strip Nuclease free water 100 pl 4 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on a Plant RNA plate 5 Start the PURE_RNAPlant_Duo protocol using the KingFisher Duo and load the plate and elution strip Thermo Scientific KingFisher Pure RNA Plant Kit 21 Chapter5 Protocols and Pipetting Instructions Switch on the KingFisher Duo making sure that you are using the Thermo Scientific KingFisher Duo 12 pin magnet head and heating block Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_RNAPlant_Duo protocol Insert the Plant RNA plate and elution strip into the instrument as indicated on the KingFisher Duo display and press OK Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user When the KingFisher Duo is to be run as a standalone instrument transfer the PURE_RNAPlant_Duo protocol to the KingFisher Duo The instructions for ransferring the protocol can be found in Chapter 4 Using the software in the Binalt Software for KingFisher Instruments version 3 2 User Manual gt 6 Add Rebinding Buffer and ethanol to row A during the dispense step When the KingFisher Duo pauses at the dispense step after the lysis step at approximately 25 minutes after starting the protocol run remove the pl
14. T THE PRODUCTS ARE ERROR FREE OR WILL ACCOMPLISH ANY PARTICULAR RESULT THERMO FISHER SCIENTIFIC HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS Exclusion of Liability Thermo Fisher Scientific and its affiliates shall have no liability to an End User arising out of the use or inability to use the product including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise Thermo Fisher Scientific Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Appendix A Appendix B Table of Contents Kit Content ocio ir i eweeter veces 5 Storage COnGIIONS eiee upeita aa dh ae riy 6 Additional Reagents Required 0 cece eee eee eee 6 Product Description 00ccee eee eee eee ee 9 INTOdUCUO socia matar indi id 9 INtENdE UsSb lt ooori io ee 9 Principle and Procedure 1 cece eects 10 Kit SPOCHICATIONS co ese op earn ood ee 10 KingFisher Magnetic Particle Processors 11 Safety Information lt lt lt lt lt lt ooo00oooeoooooomm o 13 Storage Conditions and Preparation of the Reagents 15 DMOKAGE GONTIION
15. a premix of Rebinding Buffer and ethanol if desired Plate name Added reagent volume per well DNase Rebinding Buffer 150 ul Ethanol 400 pul 6 Place the DNase plate back into the instrument and press Start After the pause the protocol will continue to completion 7 After the run is completed remove the plates and store the purified RNA When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and switch off the instrument The purified RNA is ready for use in downstream applications When working with RNA keep the purified samples on ice Store the purified RNA at 20 C or 80 C NOTE The final RNA concentration in the nuclease free water may increase if the purified RNA is eluted into a smaller than recommended volume of water but this can slightly reduce the overall RNA yield Instructions for KingFisher Duo with 12 pin Magnet Head These instructions are intended for RNA purification from 400 ul of plant lysate using the KingFisher Pure RNA Plant Kit Cat No 98060196 or 98060496 and the KingFisher Duo with 12 pin magnet head When using the KingFisher Pure RNA Plant Kit for the first time prepare the DNase storage solution Rebinding Buffer Wash Buffer 1 and Wash Buffer 2 For each run prepare the DNase working solution and Lysis Buffer with 2 M DTT For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents
16. ate from the instrument and add the Rebinding Buffer and ethanol into row A on the Plant RNA plate to rebind the RNA One can make a premix of Rebinding Buffer and ethanol if desired Added reagent Emini volume per well A DNase Rebinding Buffer 150 ul Ethanol 400 ul 7 Place the plate back into the instrument and press OK After the pause the protocol will continue to completion 8 After the run is completed remove the plate and elution strip 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 switch off the instrument The purified RNA is ready for use in downstream applications When working with RNA keep the purified samples on ice Store the purified RNA at 20 C or 80 C NOTE The final RNA concentration in the nuclease free water may increase if the purified RNA is eluted into a smaller than recommended volume of water but this can slightly reduce the overall RNA yield 22 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 Quantification and Determination of the Purity of RNA Itis 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 A One unit at 260 nm corresponds to 40 ug of RNA per ml The ratio between the
17. e 96 samples 384 samples Lysis Buffer 72 ml 2x 140 ml KingFisher Magnetic Beads 2x 1 4 ml 10 6 ml DNase lyophilized 1 vial 4 vials DNase Reconstitution Buffer 1 ml 2x1 ml 2 x DNase Buffer 12 ml 45 ml Manganese Chloride Solution 3x 1 ml 9x1ml Rebinding Buffer conc 20 ml 70 ml Wash Buffer 1 conc 125 ml 3x 125 ml Wash Buffer 2 conc 50 ml 3x50 ml Nuclease free water 30 ml 125 ml Addition of ethanol required The KingFisher Pure RNA Plant Kit Cat No 98060196 or 98060496 is intended for the purification of plant samples using the Thermo Scientific TM KingFisher Flex with a 96 deep well head or the Thermo Scientific TM KingFisher Duo with a 12 pin head and a sample volume of 50 mg of fresh plant tissue The user will need the KingFisher Flex or KingFisher Duo magnetic particle processor for conducting purification Table 1 2 In addition several common laboratory instruments and consumables are necessary to conduct an Thermo Scientific KingFisher Pure RNA Plant Kit 5 Chapter 1 Kit Content efficient purification For more details refer to Chapter 5 Protocols and Pipetting Instructions Suitable consumables for the KingFisher Duo and KingFisher Flex are listed in Table 1 3 and Table 1 4 Storage Conditions Upon arrival of the kit store the DNase DNase Reconstitution Buffer and Manganese Chloride Solution at 20 C Also after dissolving of the DNase continue to keep it at 2
18. entific T 81 Wyman Street hermo Waltham MA 02451 SCIENTIFIC Part of Thermo Fisher Scientific
19. from the lysed sample The magnetic rods will not be able to collect all the particles unless the viscose samples are diluted before the beginning of the purification process Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the Elution Buffer the addition of a small amount of detergent e g Tween 20 may improve the results 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 28 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Ordering Information Table B 1 KingFisher Pure RNA Plant Kits Cat No Product Package size 98060196 KingFisher Pure RNA Plant Kit 96 98060496 KingFisher Pure RNA Plant Kit 384 Table B 2 KingFisher Flex consumables Cat No Product Package size 97002514 KingFisher Flex 96 tip comb for PCR magnet 80 pcs 97002524 KingFisher Flex 96 tip comb for KF magnet 100 pcs 97002534 KingFisher Flex 96 tip comb for deep well magnet 100 pcs 97002610 KingFisher Flex 24 deep well tip comb and p
20. he RNA is released The volume of the Elution Buffer can be modified depending on user requirements concerning the purified total RNA concentration The final RNA concentration in the Elution Buffer may increase if the purified RNA is eluted into a smaller than recommended volume of the buffer but this can slightly reduce the overall RNA yield The modifications of the elution step must be done in Bindlt Software 3 2 and according to the volume ranges suitable for the KingFisher instrument The table below indicates the available elution volumes of the KingFisher instruments Thermo Scientific KingFisher Pure RNA Plant Kit 25 Chapter 6 General Information Table 6 1 Available elution volumes of the KingFisher Flex and KingFisher Duo Elution KingFisher instrument ER KingFisher Flex with 96 deep well head elution in a KingFisher Flex 50 150 pl 96 KF plate KingFisher Flex with 96 deep well head elution in a Microtiter deep well 50 1000 pl 96 plate ingFisher Flex with 24 deep well head 200 5000 ul K KingFisher Duo with 12 pin magnet head elution in an elution strip 30 130 pl K ingFisher Duo with 12 pin magnet head elution in a Microtiter deep 50 1000 pul well 96 plate To maximize the yield of purified 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 sedimented mag
21. late 50 pcs 97002540 KingFisher Flex 96 KF plate 200 ul 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs Thermo Scientific KingFisher Pure RNA Plant Kit 29 Appendix B Ordering Information Table B 3 KingFisher Duo consumables Cat No Product 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 24 48 pcs deep well plate 97003520 KingFisher Duo elution strip 40 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples 30 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific www thermoscientific com 2013 Thermo Fisher Scientific Inc All rights reserved All trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details Thermo Fisher Sci
22. netic bead pellet s 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 26 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Troubleshooting Problem Possible cause and actions Low RNA yield 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 Efficient homogenization of the plant samples increases the total RNA yield Prolonged storage of the sample material may reduce the total RNA yield Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of products from other manufacturers may cause unsuitable mixing and affect the yield of purified 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 eluted RNA Continued Thermo Fisher Scientific Thermo Scientific KingFisher Pure RNA Plant Kit 27 Appendix A Troubleshooting Cont Possible cause and actions Magnetic particles remaining in the sample or elution well Starting material that is too viscose prevents efficient collection of the KingFisher Magnetic Beads
23. red through the wash steps Two different Wash Buffers are used followed by an air drying step High quality RNA is eluted into the Elution Buffer and is ready for subsequent downstream processes s Kit Specifications The KingFisher Pure RNA Plant Kit is designed for rapid automated preparation of highly pure total RNA from plant samples using KingFisher magnetic particle processors If a dispense step requiring the addition of the Binding Buffer is excluded the approximate processing time is 60 minutes for the purification of 96 samples on the KingFisher Flex and 12 samples on the KingFisher Duo The obtained RNA can be used directly in various downstream applications Fresh or frozen plant samples can be used A suitable amount of fresh material is 20 50 mg Use of young plant samples and or if possible keeping plants for 12 h in darkness before collecting the samples reduces the polysaccharide and polyphenolic contents which may interfere in downstream applications Suitable sample storage as well as an efficient homogenization step is essential for obtaining a high yield and good quality total RNA Typically 5 50 ug of total RNA can be purified from 50 mg of fresh plant sample with an A A ratio of gt 1 8 2 1 The yields of acquired purified RNA depend on the sample type and the method of sample collection storage and disruption 10 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Product
24. rotiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples Thermo Fisher Scientific Thermo Scientific KingFisher Pure RNA Plant Kit 7 Thermo Fisher Scientific Product Description Introduction The KingFisher Pure RNA Plant Kit is designed for rapid automated purification of RNA from plant samples using Thermo Scientific TM KingFisher instruments The RNA purified using the KingFisher Pure RNA Plant Kit is of high quality and free of proteins nucleases and other contaminants or inhibitors It is therefore suitable for direct use in many different downstream applications such as RT qPCR reverse transcription quantitative PCR RT PCR and several other enzymatic reactions Intended Use The KingFisher Pure RNA Plant Kit is developed for purification of total RNA from plant samples using paramagnetic particles The reagents and specific plastic consumables are designed for use with the KingFisher Flex and KingFisher Duo magnetic particle processors as part of an integrated system The KingFisher Pure RNA Plant Kit enables extraction of RNA from fresh samples or samples frozen immediately after collection and stored at 80 C The KingFisher Pure RNA Plant Kit is only intended for research use not for clinical or diagnostic use The user is responsible for validating the performance of the Thermo Scientific KingFisher instrument and the KingFisher Pure RNA Plant Kit for any
25. the KingFisher Flex with 96 deep well plates When using the KingFisher Pure RNA Plant Kit for the first time prepare the DNase storage solution Rebinding Buffer Wash Buffer 1 and Wash Buffer 2 For each run prepare the DNase working solution and Lysis Buffer with 2 M DIT For more instructions refer to Chapter 4 Storage Conditions and Preparation of the Reagents Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 C 1 Take six empty Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates 18 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 2 Fill the plates as follows Plate Sample reagent mm Plate type Plate name Content volume per well 1 Microtiter deep Sample Plant Iysate 400 ul well 96 plate KingFisher 25 pl Magnetic Beads Ethanol 400 pi 2 Wash 1_1 Wash Buffer 1 900 u DNase DNase 200 u working solution 4 Wash 1_2 Wash Buffer 1 700 pi 5 Wash 2_1 Wash Buffer 2 700 u 6 Wash 2_2 Wash Buffer2 700 u 7 KingFisher Flex Elution Nuclease free 100 y 96 KF plate water Resuspend the KingFisher Magnetic Beads well by vortexing before use 3 Place a Thermo Scientific King
26. tial step before RNA purification in order to gain a good yield of high quality RNA Plant tissue can be homogenized for example with a pestle using steel beads or with commercial homogenizers of which high throughput homogenizers provide a suitable method for handling 96 samples simultaneously The homogenization step must disrupt the structures of the starting material rapidly and completely in order to ensure a high yield of RNA When purifying RNA from woody lignified and or polyphenol rich samples such as branches twigs needles wax coated leaves e g laurel and wheat flour supplement the Lysis Buffer with polyvinylpyrrolidone PVP at a 2 w v final concentration Homogenize 20 50 mg of fresh plant sample Follow the instructions of the available homogenization tool Add before or after homogenization depending on the system 600 ul of Lysis Buffer including 2 M DTT to each sample Incubate the sample at 56 C for 5 min To clear the plant lysate centrifuge the sample at 20 000 x g for 10 min Transfer 400 ul of cleared supernatant to a Thermo Scientific Microtiter deep well 96 plate and begin the purification using the KingFisher Flex or KingFisher Duo Refer to the detailed instructions below Instructions for KingFisher Flex with 96 Deep Well Plates These instructions are intended for RNA purification from 400 ul of plant lysate using the KingFisher Pure RNA Plant Kit Cat No 98060196 or 98060496 and
27. tion manual It is recommended that Good Laboratory Practice GLP is followed to guarantee reliable analyses Table 3 1 Safety precautions Reagent Hazardous contents Safety instructions Lysis Buffer Guanidium thiocyanate Harmful by inhalation in contact Sodium N layroylsarcosinate with skin and if swallowed Liberates very toxic gas in contact with acids Harmful to aquatic organisms and may cause long term adverse effects in the aquatic environment Keep the container in a well ventilated place Do not breathe gas fumes vapor spray Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste Avoid release to the environment Refer to special instructions safety data sheets Continued Thermo Fisher Scientific Thermo Scientific KingFisher Pure RNA Plant Kit 13 Chapter 3 Safety Information Cont Hazardous contents Safety instructions Rebinding Buffer Guanidium chloride Harmful if swallowed Irritating to conc eyes and skin Do not breathe gas fumes vapor spray In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste Wash Buffer 1 Guanidium chloride Harmful if swallowed Irritating to conc eyes and skin Do not breathe gas fumes vapor spray In case of contact with e
28. yes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste 14 Thermo Scientific KingFisher Pure RNA Plant Kit Thermo Fisher Scientific Storage Conditions and Preparation of the Reagents Storage Conditions Upon arrival of the kit store the DNase DNase Reconstitution Buffer and Manganese Chloride Solution at 20 C Also after dissolving of the DNase continue to keep it at 20 C Store the KingFisher Magnetic Beads at 4 C Other kit components can be stored at room temperature 15 25 C The reagents are stable for up to two years from the manufacturing date Preparation of the Lysis Buffer Before each RNA purification a fresh aliquot of Lysis Buffer should be supplemented with DTT not provided Calculate the amount of Lysis Buffer needed It is required to use 600 ul of Lysis Buffer for the purification of one sample Add 20 ul of 2 M DTT to each 1 ml of Lysis Buffer Preparation of the Wash Buffers Add 96 100 ethanol to the concentrated Wash Buffer 1 and Wash Buffer 2 as indicated below in Table 4 1 prior to the first use Thermo Fisher Scientific Thermo Scientific KingFisher Pure RNA Plant Kit 15 Chapter 4 Storage Conditions and Preparation of the Reagents Table 4 1 Instructions for the preparation of Wash Buffer 1 and Wash Buffer 2 Add the indicated volume of
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