KingFisher Pure DNA Plant Kit Instruction Manual
Contents
1.2. KingFisher Flex KingFisher Duo 96 formats 24 format 12 format 6 format ae 20 1000 u 200 5000 ui 50 1000 pi 200 5000 pi volume Capacity Up to 96 Up to 24 Upto 12 Up to 6 samples per samples per samples per samples per run sample run sample run sample run sample volume 200 ul volume 1 ml volume 200 ul volume 1 ml Magnetic head 96 inter 24 format for 12 pin magnet 6 pin magnet changeable KingFisher Flex head for head for formats for 24 deep well Microtiter deep KingFisher Flex PCR plate plate well 96 plate 24 deep well KingFisher Flex plate 96 KF plate Microtiter deep well 96 plate Plates KingFisher Flex KingFisher Flex Microtiter deep KingFisher Flex 96 KF plate 24 deep well well 96 plate 24 deep well 20 200 u plate 50 1000 ul plate 96 well PCR 200 5000 ul KingFisher Duo 200 5000 pul plate skirted elution strip 20 100 ul 30 130 ul Microtiter deep well 96 plate 50 1000 ul 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 abov
3. Thermo Scientific KingFisher Pure DNA Plant Kit Revision 1 0 N14038 March 2013 hermo Copyright 2013 Thermo Fisher Scientific Inc All rights reserved Tween is a trademark of ICI Americas Inc Virkon is a trademark of E l du Pont de Nemours and Company or its affiliates All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries Reproduction of the accompanying user documentation in whole or in part is prohibited Disclaimer Thermo Fisher Scientific reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without prior notice as part of continuous product development Although this manual has been prepared with every precaution to ensure accuracy Thermo Fisher Scientific assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information This instruction manual supersedes all previous editions Products are for Research Use Only Not for use in diagnostic procedures The Product will operate substantially in conformance with Thermo Fisher Scientific s published specifications THERMO FISHER SCIENTIFIC DISCLAIMS ALL OTHER WARRANTIES WHETHER EXPRESSED OR IMPLIED ORAL OR WRITTEN WITH RESPECT TO THE PRODUCTS INCLUDING WITHOUT LIMITATION ALL IMPLIED WARRANTIES OF PRODUCT QUALITY CONDITION DESCRIPTION MERCHANTABILITY OR FITNESS FOR ANY PAR
4. 15 Storage GonditionS u una nennen aS 15 Preparation of the Wash Buffers 2 22 2222 cece eee eae 15 Preparation of the Lysis BufferA 0 cece eee eee 16 Protocols and Pipetting Instructions 17 Handling of KingFisher Magnetic BeadS 17 Sample Material and Storage 0 ee eee 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 and Deep Well 96 Plato coo ceo ar 20 Quantification and Determination of the Purity of DNA 22 General Information 00cceeee eee eee eee 23 Reagent Specificity and VOluMES o o ooooom 23 Handling of Magnetic Beads 0 cece eee eee 23 Binding Wash and Elution Steps 0 00 oo 23 Troubleshooting 000cce cece eee e eee e eee eeee 25 Ordering Information oooooocoororoan nenn 27 NOTE For more details on storing the kit reagents refer to Storage Conditions on page 6 Thermo Scientific KingFisher Pure DNA Plant Kit 3 Thermo Fisher Scientific Kit Content Table 1 1 Thermo Scientific KingFisher Pure DNA Plant Kit Cat No 98050196 98050496 Package size 96 samples 384 samples Lysis Buffer A 60 ml 225 ml Lysis Buffer B 8 4 ml 32 ml Precipitation Solution 16 5 ml 60 ml KingFisher Magnetic Beads
5. Additional Reagents Required e 96 100 ethanol EtOH molecular biology grade e Dithiothreitol DTT for seeds 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 DNA 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 Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Plant Kit 7 Thermo Fisher Scientific
6. Bindlt Software 3 2 can be used for running ready made and optimized protocols for the Thermo Scientific KingFisher Pure Kits It is also possible to transfer the developed protocol onto the onboard software and run it directly from the instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting in high purity DNA without the risk of carryover or cross contamination The KingFisher instrument family comprises four systems covering working volumes from 20 to 5000 ul Each system consists of an instrument specially designed plastic consumables and the easy to use Bindlt Software 3 2 The KingFisher Pure DNA Plant Kit is optimized and ready for use with the KingFisher Flex or KingFisher Duo 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 DNA Plant Kit are available for the KingFisher Flex 96 and KingFisher Duo For more information go to www thermoscientific com kingfisherinfo or contact your local authorized distributor Thermo Scientific KingFisher Pure DNA Plant Kit 11 Chapter 2 Product Description Table 2 1 Overview of KingFisher Flex and KingFisher Duo magnetic particle processors
7. Product Description Introduction The KingFisher Pure DNA Plant Kit is designed for rapid automated purification of DNA from lt 50 mg of plant material using Thermo Scientific KingFisher instruments The DNA purified using the KingFisher Pure DNA 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 PCR polymerase chain reaction restriction analysis and several other enzymatic reactions Intended Use The KingFisher Pure DNA Plant Kit is developed for purification of DNA from plant material 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 DNA Plant Kit is suitable for extraction of DNA from fresh or frozen plant material The KingFisher Pure DNA 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 DNA Plant Kit for any particular use as the performance of the kits has not been validated for any specific species or downstream application Principle and Procedure The KingFisher Pure DNA Plant Kit uses magnetic particle technology for DNA purification The Thermo Sc
8. and storage conditions With dried material use only one fifth of the starting material compared to the fresh sample material Homogenization of Sample Material Efficient homogenization of the sample material is an essential step before DNA purification in order to gain a good yield of high quality DNA 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 DNA When purifying DNA 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 A with polyvinylpyrrolidone PVP at a 2 w v final concentration When purifying DNA from seeds e g Brassica napus supplement the Lysis Buffer A with dithiothreitol DTT at a 40 mM final concentration Homogenize lt 50 mg of fresh plant sample or up to 10 mg if dried samples are used After homogenization add 350 ul of Lysis Buffer A to a sample and vortex for 20 s Add 50 ul of Lysis Buffer B to the sample If RNase A reatment is needed to reduce the amount of RNA in the sample add 20 ul of RNase A to each sample RNase A treatment is recommended for the samples containing large amounts of RNA
9. avoid the risk of transferring variable amounts of the beads to the respective wells The amount of beads in the wells affects the yield of the purified DNA Binding Wash and Elution Steps The binding between the purified DNA and the KingFisher Magnetic Beads is strong in the presence of a chaotropic salt The binding will remain throughout the wash steps until the elution where the DNA is released The volume of the Elution Buffer can be modified depending on user requirements concerning the purified DNA concentration The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of the buffer but this can slightly reduce the overall DNA yield The modifications of the elution step must be done in Bindlt Software 3 2 and according to the volume ranges suitable for the KingFisher instrument The table below indicates the available elution volumes of the KingFisher instruments Thermo Scientific KingFisher Pure DNA Plant Kit 23 Chapter 6 General Information Table 6 1 Available elution volumes of the KingFisher Flex and KingFisher Duo rn A 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 KingFisher Flex with 24 deep well head 200 5000 ul KingFisher Duo with 12 pin magnet head el
10. 2x 1 4 ml 10 6 ml RNase A 2x1 4 ml 7x1 2 ml Wash Buffer 1 conc 50 ml 2x50 ml Wash Buffer 2 conc 60 ml 3x 60 ml Elution Buffer 30 ml 70 ml Addition of ethanol required The KingFisher Pure DNA Plant Kit Cat No 98050196 or 98050496 is intended for the purification of genomic DNA from plant material using the Thermo Scientific KingFisher Flex with a 96 deep well head or the Thermo Scientific KingFisher Duo with a 12 pin head and a sample amount of up to 50 mg 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 efficient purification For more details refer to Chapter 5 Protocols and Pipetting Instructions Suitable consumables for the KingFisher Duo and KingFisher Flex are listed in Table 1 3 and Table 1 4 Thermo Scientific KingFisher Pure DNA Plant Kit 5 Chapter 1 Kit Content Storage Conditions Upon arrival store the Thermo Scientific KingFisher Magnetic Beads at 4 C All the other buffers and reagents included in the KingFisher Pure DNA Plant Kit can be stored at room temperature 15 25 C The RNase A solution is stable at room temperature until the seal of the vial is broken After being opened the vial should be stored at 20 C The reagents are stable for up to three years from the manufacturing date
11. Vortex the samples for 20 s and incubate at 65 C for 10 min with occasional vortexing or shaking To clear the plant lysate add 130 ul of Precipitation Solution and mix by inverting the sample 3 times followed by incubation on ice for 5 min Centrifuge the sample for 5 min 20 000 x g Transfer 400 ul of supernatant o a Thermo Scientific Microtiter deep well 96 plate and begin the purification of DNA using the KingFisher Flex or KingFisher Duo Refer to he detailed instructions below Instructions for KingFisher Flex with 96 Deep Well Plates These instructions are intended for DNA purification from 400 ul of plant lysate using the KingFisher Pure DNA Plant Kit Cat No 98050196 or 98050496 and the KingFisher Flex with Microtiter deep well 96 plates 18 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 When using the KingFisher Flex and the KingFisher Pure DNA Plant Kit for the first time prepare the Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Preparation of the Wash Buffers on page 15 Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the solution at 37 C and equilibrate to room temperature 15 25 0 1 Homogenize the samples according to the instructions in Homogenization of Sample Material on page 18 2 Take four empt
12. a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield Quantification and Determination of the Purity of DNA It is recommended to measure the absorbance at 320 nm 280 nm and 260 nm The concentration of DNA can be defined with the absorbance at 260 nm A One unit at 260 nm corresponds to 50 ug of DNA per ml The ratio between the A __ A indicates the purity of the DNA The value for DNA 260 280 should be gt 1 7 2 0 It is recommended that A correction is used for the absorbance values Subtract the A from the A and A ratios to remove the effects of carryover of the magnetic particles A 260 00 e Total amount of DNA isolated concentration x volume of sample in ml e Purity of DNA sample Ajo As20 Aso A e Concentration of DNA sample 50 pg ml x A X dilution factor 260 00 22 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific General Information Reagent Specificity and Volumes A reagent must not be used with any kit other than that for which it is intended It is strongly recommended that the volume of reagents in each well is kept within the limits specified in the KingFisher Flex User Manual or the KingFisher Duo User Manualto avoid spillover and to maximize efficiency of performance Handling of Magnetic Beads The KingFisher Magnetic Beads should be mixed thoroughly before use to
13. tip comb into row B on a Plant DNA plate 5 Start the PURE_DNAPlant_Duo protocol using the KingFisher Duo and load the plate and elution strip Switch on the KingFisher Duo making sure that you are using the Thermo Scientific KingFisher Duo 12 pin magnet head and heating block Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Plant Kit 21 Chapter 5 Protocols and Pipetting Instructions Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_DNAPlant_Duo protocol Insert the Plant DNA plate and elution strip into the instrument as indicated on the KingFisher Duo display and press OK Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user When the KingFisher Duo is to be run as a standalone instrument transfer he PURE_DNABlood_Duo protocol to the KingFisher Duo The instructions for transferring the protocol can be found in Chapter 4 Using the software n the Binalt Software for KingFisher Instruments version 3 2 User Manual s 6 When the protocol is completed remove the plate and elution strip according to the instructions on the KingFisher Duo display and switch off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into
14. TICULAR PURPOSE THERMO FISHER SCIENTIFIC DOES NOT WARRANT THAT THE PRODUCTS ARE ERROR FREE OR WILL ACCOMPLISH ANY PARTICULAR RESULT THERMO FISHER SCIENTIFIC HEREBY EXPRESSLY DISCLAIMS ANY WARRANTY REGARDING RESULTS OBTAINED THROUGH THE USE OF THE PRODUCTS INCLUDING WITHOUT LIMITATION ANY CLAIM OF INACCURATE INVALID OR INCOMPLETE RESULTS Exclusion of Liability Thermo Fisher Scientific and its affiliates shall have no liability to an End User arising out of the use or inability to use the product including without limitation for any loss of use or profits business interruption or any consequential incidental special or other indirect damages of any kind regardless of how caused and regardless of whether an action in contract tort strict product liability or otherwise Thermo Fisher Scientific Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Appendix A Appendix B Table of Contents Kit Content i eweeter veces 5 Storage COnGIIONS cocoa ca ae riy 6 Additional Reagents Required 0 cece eee eee eee 6 Product Description 000ccee cece cence eee eens 9 INFODUCHON NO 9 IntendediUse 4 2 00 rer 9 Principle and Procedure 0 cee eee cece een 9 Kit SPOCHICATIONS lt 4 esi op ecto de ee 10 KingFisher Magnetic Particle Processors 10 Safety Information 20000cc cece eee eee eens 13 Storage Conditions and Preparation of the Reagents
15. e on the Plates row 12 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Safety Information The following components of the KingFisher Pure DNA 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 instruction manual It is recommended that Good Laboratory Practice GLP is followed to guarantee reliable analyses Table 3 1 Safety precautions Reagent Hazardous contents Safety instructions Wash Buffer 1 conc Guanidinium chloride Harmful if swallowed Irritating to eyes and skin Do not breathe gas fumes vapor spray In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves This material and its container must be disposed of as hazardous waste Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Plant Kit 13 Storage Conditions and Preparation of the Reagents Storage Conditions Upon arrival store the KingFisher Magnetic Beads at 4 C All the other buffers and reagents included in the KingFisher Pure DNA Blood Kit can be stored at room temperature 15 25 C The Proteinase K solution is stable at room temperature until the seal of the vial is broken After being opened the vial should be stored at 20 C The reagents are stable for up to three years from the manufactur
16. ffer If the KingFisher magnetic particle processor does not work properly refer to the relevant user manual of the KingFisher instrument in use Inhibition of downstream enzymatic reactions If the purified DNA contains residual salt use the correct order for the Wash Buffers Always wash the KingFisher Magnetic Beads with Wash Buffer 1 first and then proceed with Wash Buffer 2 26 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Ordering Information Table B 1 KingFisher Pure DNA Plant Kits Cat No Product Package size 98050196 KingFisher Pure DNA Plant Kit 96 98050496 KingFisher Pure DNA 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 plate 50 pcs 97002540 KingFisher Flex 96 KF plate 200 ul 48 pcs 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs Thermo Scientific KingFisher Pure DNA Plant Kit 27 Appendix B Ordering Information Table B 3 KingFisher Du
17. ientific KingFisher technology combines the speed and efficiency of DNA purification with easy handling of magnetic Thermo Scientific KingFisher Pure DNA Plant Kit 9 Chapter 2 Product Description particles The purification process requires no phenol chloroform extraction and needs very little hands on time The first step of the protocol binds the DNA from the homogenized plant material to the surface of the KingFisher Magnetic Beads in the presence of the Binding Buffer The KingFisher Magnetic Beads are highly reactive superparamagnetic beads The following three effective wash steps dispose of proteins cell debris and any residual contaminants while the DNA is bound to the KingFisher Magnetic Beads and transferred through the wash steps Two different Wash Buffers are used followed by an air drying step which improves the purity of the DNA High quality DNA is eluted into the Elution Buffer and is ready for subsequent downstream processes Kit Specifications The KingFisher Pure DNA Plant Kit is designed for rapid automated preparation of highly pure DNA from plant material using KingFisher magnetic particle processors If the homogenization of the starting material is excluded the approximate processing time is 40 minutes for the purification of 96 samples on the KingFisher Flex and 12 samples on the KingFisher Duo when the sample volume is 400 ul The obtained DNA can be used directly in various downstream applicatio
18. ing date Preparation of the Wash Buffers Add the indicated volume of isopropanol 100 and ethanol 96 100 to Wash Buffer 1 and Wash Buffer 2 as indicated below in Table 4 1 prior to the first use Table 4 1 Instructions for the preparation of the buffers Add the indicated volume per bottle 96 samples Cat No 98050196 and 384 samples Cat No 98050496 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 50 ml 60 ml Ethanol 96 100 150 ml 180 ml Total volume 200 ml 240 ml After preparing each solution mark the bottle to indicate that the step has been completed The buffers can be stored at room temperature Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Plant Kit 15 Chapter 4 Storage Conditions and Preparation of the Reagents Preparation of the Lysis Buffer A When purifying DNA 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 A with polyvinylpyrrolidone PVP at a 2 w v final concentration When purifying DNA from seeds e g Brassica napus supplement the Lysis Buffer A with dithiothreitol DTT at a 40 mM final concentration 16 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Before beginning the DNA purification protocol carefully read through the Therm
19. lution at 37 C and equilibrate to room temperature 15 25 C 1 Homogenize the samples according to the instructions in Homogenization of Sample Material on page 18 2 Prepare the Plant DNA plate i e a Microtiter deep well 96 plate 20 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 Add the following reagents to the rows Note that row B is reserved for the tip comb and should be left empty Note that rows C D and E are also left empty Sample reagent name and Row Rpw name Content volume per well Plant DNA A Sample Homogenized 400 ul plate supernatant Microtiter KingFisher 25 pil deep well Magnetic 96 plate Beads Ethanol 400 ul B Tip 12 tip comb Empty c Empty Empty Empty D Empty Empty Empty E Empty Empty Empty F Wash 1 Wash Buffer 1 900 ul G Wash 2_1 Wash Buffer 2 800 ul H Wash 2_2 Wash Buffer 2 800 ul 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 Elution Buffer is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Elution Buffer 100 yl 4 Place a Thermo Scientific KingFisher Duo 12
20. ns Fresh or frozen plant material equaling lt 50 mg can be used The procedure is optimized for sample volumes of 400 ul Depending on the sample up to 15 ug of DNA can be purified from 50 mg of fresh plant material with an A A ratio of gt 1 7 2 0 The DNA yields depend on the sample type sample storage and homogenization method KingFisher Magnetic Particle Processors The KingFisher magnetic particle processors are designed for the automated transfer and processing of magnetic particles in microplate format The patented technology of the Thermo Scientific KingFisher systems is based on the use of magnetic rods covered with a disposable specially designed tip comb and plates or tubes Use only Thermo Scientific KingFisher plastic consumables as use of products from other manufacturers may cause unsuitable mixing or even instability in the KingFisher instrument The instrument functions without any dispensing or aspiration parts or devices Samples and reagents including magnetic particles are dispensed onto the plates according to the corresponding instructions Dispensing can be carried out manually or partially automatically using automatic 10 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Product Description Chapter 2 dispensers for example the Thermo Scientific MultidropTM Combi and or the Thermo Scientific Versette Thermo Scientific
21. o 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 Binalt Software for KingFisher Instruments version 3 2 User Manual Cat No N07974 Bindlt Software 3 2 protocols can be found at www thermoscientific com kingfisher Handling of KingFisher Magnetic Beads A homogeneous distribution of the KingFisher Magnetic Beads in the container is essential before the beads are transferred to the wells in order to ensure a high consistency between the wells To gain complete resuspension of the beads shake the container vigorously or vortex briefly The paramagnetic beads have a tendency to sediment relatively quickly Sample Material and Storage Use lt 50 mg of fresh or frozen plant material as sample material To minimize DNA degradation avoid repeated freezing and thawing of the samples Using 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 Appropriate sample storage is essential for reproducibility and high DNA Thermo Scientific KingFisher Pure DNA Plant Kit 17 Chapter 5 Protocols and Pipetting Instructions yields The yields of DNA may vary depending on sample age type of sample
22. o consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 48 pcs 24 deep well plate 95040450 Microtiter deep well 96 plate 50 pcs 95040460 Microtiter deep well 96 plate sterile 50 pcs 97003520 KingFisher Duo elution strip 40 pcs 95040470 KingFisher Flex 24 deep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples 28 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Notes Thermo Fisher Scientific Thermo Scientific KingFisher Pure DNA Plant Kit 29 Notes 30 Thermo Scientific KingFisher Pure DNA 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 Scientific T 81 Wyman Street hermo Waltham MA 02451 SCIENTIFIC Part of Thermo Fisher Scientific
23. products from other manufacturers may cause unsuitable mixing and affect the yield of purified DNA Low purity RNA contamination Insufficient washing causes impurities in the Elution Buffer Carry out the RNase A treatment step described in the purification procedure Continued Thermo Scientific KingFisher Pure DNA Plant Kit 25 Appendix A Troubleshooting Cont Possible cause and actions Magnetic particles remaining in the sample or elution well Starting material that is too viscose prevents efficient collection of the KingFisher Magnetic Beads from the lysed sample The magnetic rods will not be able to collect all the particles unless the viscose samples are diluted before the beginning of the purification Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the Elution Buffer the addition of a small amount of detergent e g Tween 20 may improve the results Alternatively centrifuge the eluates or place them on a magnet for a few minutes to collect the residual beads at the bottom of the well Carryover of the KingFisher Magnetic Beads does not affect most downstream processes KingFisher Magnetic Beads that occasionally remain 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 Bu
24. splay 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_DNAPlant_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 2 User Manual 6 When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and switch off the instrument Store the purified DNA accordingly The purified DNA is ready for use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified DNA is eluted into a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield Instructions for KingFisher Duo with 12 pin Magnet Head and Deep Well 96 Plate These instructions are intended for DNA purification from 400 ul of plant lysate using the KingFisher Pure DNA Plant Kit Cat No 98050196 or 98050496 and the KingFisher Duo with Microtiter deep well 96 plates When using the KingFisher Duo and the KingFisher Pure DNA Plant Kit for the first time prepare the Wash Buffer 1 and Wash Buffer 2 For more instructions refer to Preparation of the Wash Buffers on page 15 Check all the solutions in the kit for salt precipitation before each use Redissolve precipitates by warming the so
25. ution in an elution strip 30 130 pl KingFisher Duo with 12 pin magnet head elution in a Microtiter deep 50 1000 pul well 96 plate To maximize the yield of purified DNA avoid 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 magnetic beads should be completely resuspended In addition the volume of the Elution Buffer should be adequate for efficient mixing of the beads in order to obtain a maximal release of the purified DNA from the beads 24 Thermo Scientific KingFisher Pure DNA Plant Kit Thermo Fisher Scientific Thermo Fisher Scientific Troubleshooting Problem Possible cause and actions Low DNA yield Insufficient homogenization of plant material To disrupt the cell wall it is important to homogenize the sample thoroughly Make sure that ethanol was added to Wash Buffer 1 and Wash Buffer 2 before use Follow the instructions for Wash Buffer preparation on page 15 Do not let the KingFisher Magnetic Beads dry as this may result in lower elution efficiency Prolonged storage or repeated freeze thaw cycles of the sample material may reduce the DNA yield There should be an adequate volume of the Elution Buffer to cover the KingFisher Magnetic Beads completely during the elution step Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of
26. y Microtiter deep well 96 plates and two Thermo Scientific TM KingFisherTM Flex 96 KF plates Add the following reagents to the Sample plate and leave the plate at room temperature while the other plates are being filled 3 Fill the plates as follows Plate number Reagent volume per well Plate type Plate name Content Microtiter deep well 96 plate Plant lysate KingFisher 25 ul Magnetic Beads Ethanol 400 ul Wash 1 Wash Buffer 1 900 ul Wash 2_1 Wash Buffer 2 800 ul Wash 2_2 Wash Buffer 2 800 ul KingFisher Flex Elution Elution Buffer 150 pl 96 KF plate Resuspend the KingFisher Magnetic Beads well by vortexing before use 4 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate i e an empty KingFisher Flex 96 KF plate 5 Start the PURE_DNAPlant_Flex96 protocol with 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 Thermo Scientific KingFisher Pure DNA Plant Kit 19 Chapter 5 Protocols and Pipetting Instructions Connect the PC with Bindlt Software 3 2 to the KingFisher Flex Start the PURE_DNAPlant_Flex96 protocol Insert the Tip Plate and the filled plates into the instrument as indicated on the KingFisher Flex di
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