Thermo Scientific KingFisher Pure Plasmid Kit
Contents
1. Continued 28 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Troubleshooting Appendix A Cont Possible cause and actions Purified sample Plasmid DNA denatured during cell lysis Denatured plasmid remaining in the sample or elution well contains additional DNA migrates ahead of supercoiled DNA and is not suitable for plasmid forms enzymatic manipulations such as restriction digestion To avoid denaturation do not allow the cell lysis to proceed for more than 2 min Magnetic particles 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 Improper lysis may also cause problems collecting the KingFisher Magnetic Beads If the KingFisher Magnetic Beads are inefficiently collected from the elution step 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 plasmid DNA yield as the DNA has already been released from the KingFisher Magnetic Beads into the Elution Buffer If the KingFisher magnetic particle processor does not work properly refer to the relevant user manual of the KingFi2. Thermo Scientific KingFisher Pure Plasmid Kit Revision 1 0 N14041 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 I 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 PU
3. Pure Kits It is also possible to transfer the developed protocol onto the onboard software and run it directly from the instrument The KingFisher instruments provide a rapid and automated solution for complicated and time consuming purification processes resulting in high purity DNA without risk of carryover or cross contamination Su 09 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 Plasmid 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 Plasmid Kit are available for the KingFisher Flex 96 and 24 and KingFisher Duo For more information go to www thermoscientific com kingfisherinfo or contact your local authorized distributor Thermo Fisher Scientific Thermo Scientific KingFisher Pure Plasmid Kit 11 Chapter 2 Product Description Table 2 1 Overview of KingFisher Flex and KingFisher Duo magnetic particle processors KingFisher Flex KingFisher Duo 96 deep well 24 format 12 format 6 forma
4. 12 Thermo Scientific KingFisher Pure Plasmid Ki Thermo Fisher Scientific Thermo Fisher Scientific Safety Information The following components of the KingFisher Pure Plasmid 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 Lysis Buffer Hazardous contents Sodium hydroxide Sodium dodecyl sulphate Safety instructions Causes severe burns When using do not eat or drink Do not breathe gas fumes vapor spray In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing gloves and eye face protection In case of accident or if you feel unwell seek medical advice immediately This material and its container must be disposed of as hazardous waste Continued Thermo Scientific KingFisher Pure Plasmid Kit 13 Chapter 3 Safety Information Cont Hazardous contents Safety instructions Wash Buffer 1 Guanidinium 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 m
5. s wits 2 504 200 AREER Ree EAS 15 Preparation of the Resuspension Solution 15 Preparation of the Binding Buffer and Wash Buffers 15 Protocols and Pipetting Instructions 17 Handling of KingFisher Magnetic BeadS 17 Instructions for KingFisher Flex with 96 Deep Well Plates 17 Instructions for KingFisher Duo with 12 pin Magnet Head 20 Quantification and Determination of the Purity of DNA 23 General Information 22000ceeeeeee eee eees 25 Reagent Specificity and VoluMES o ooooooo o o o o 25 Handling of Magnetic Beads n annaa eee eee 25 Binding Wash and Elution Steps o0oooo o o o 25 Decontamination and Disinfection of Sample Material 26 Troublesho0HMO iio rr 27 Ordering Information ooooooororrrron nenn 31 NOTE For more details on storing the kit reagents refer to Storage Conditions on page 6 Thermo Scientific KingFisher Pure Plasmid Kit 3 Thermo Fisher Scientific Kit Content Table 1 1 Thermo Scientific KingFisher Pure Plasmid Kit Cat No 98080196 98080496 Package size 96 samples 384 samples RNase A 0 28 ml 1ml Resuspension Solution 25 ml 90 ml Lysis Buffer 25 ml 90 ml Neutralization Solution 25 ml 90 ml KingFisher Magnetic Beads 2x 1 4 ml 10 6 ml Wash Buffer 1 conc 110 ml 3x110 ml Wash Buffer 2 conc 60 ml 3x60 ml
6. Elution Buffer 18 ml 45 ml Addition of ethanol and or isopropanol required The KingFisher Pure Plasmid Kit Cat No 98080196 or 98080496 is intended for the purification of plasmid DNA using the Thermo Scientific TM KingFisher Flex with a 96 deep well head or the Thermo Scientific M KingFisherTM Duo with a 12 pin head from an overnight coli culture 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 Plasmid Kit 5 Chapter 1 Kit Content Storage Conditions Upon arrival of the kit store the Thermo Scientific KingFisher Magnetic Beads at 4 C The RNase A solution is stable at room temperature as long as the vial remains sealed After being opened it should be stored at 20 C After the addition of RNase A the Resuspension Solution is stable for six months when stored at 4 C Other components of the kit should be stored at room temperature 15 25 C The reagents are stable for up to three years from the manufacturing date Additional Reagents Required e 96 100 ethanol EtOH molecular b
7. RPOSE 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 IO 2 00 rer 9 Principle and Procedure oooocccccocoo ooo 9 Kit SPOCHICATIONS lt 4 esi op ecto de ee 10 KingFisher Magnetic Particle Processors 10 Safety Information 20000 0c ces seen eee eeees 13 Storage Conditions and Preparation of the Reagents 15 MOKAGE GONTIIONS
8. apter 5 number Plate type Plate name Content Reagent volume 1 Microtiter deep Sample Bacterial cell 500 ul well 96 plate lysate Isopropanol 250 ul KingFisher 25 pul Magnetic Beads Resuspend the KingFisher Magnetic Beads well by vortexing before use 7 Take four empty Microtiter deep well 96 plates and an empty Thermo Scientific KingFisher Flex 96 KF plate and fill them as follows Plate number Plate type Microtiter deep well 96 plate Wash 1_1 Plate name Content Wash Buffer 1 Reagent volume Wash 1_2 Wash 2_1 Wash Buffer 1 Wash Buffer 2 Wash 2_2 Wash Buffer 2 700 pl oja Aj KingFisher Flex Elution Elution Buffer 100 yl 96 KF plate 8 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 9 Start the PURE_Plasmid_Flex96 protocol using the KingFisher Flex 96 and load the plates 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_Plasmid_Flex96 protocol Insert the Tip Plate and the filled plates into the instrument as instructed on the KingFisher Flex display After all the plates have been loaded into the instrument the protocol will star
9. aterial and its container must be disposed of as hazardous waste 14 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Storage Conditions and Preparation of the Reagents Storage Conditions Upon arrival of the kit store the KingFisher Magnetic Beads at 4 C The RNase A solution is stable at room temperature as long as the vial remains sealed After being opened it should be stored at 20 C After the addition of RNase A the Resuspension Solution is stable for six months when stored at 4 C Other components of the kit should be stored at room temperature 15 25 C The reagents are stable for up to three years from the manufacturing date Preparation of the Resuspension Solution Add the RNase A solution included in the kit to the Resuspension Solution and mix thoroughly After the addition of RNase A the Resuspension Solution is stable for six months when stored at 4 C For longer storage aliquot the Resuspension Solution into an appropriate number of aliquots and supplement one aliquot with 10 ul of RNase A per 1 ml of Resuspension Solution Store the remaining RNase A at 20 C Preparation of the Binding Buffer and Wash Buffers Add isopropanol and 96 100 ethanol to concentrated Wash Buffer 1 and Wash Buffer 2 as indicated below in Table 4 1 prior to the first use Thermo Scientific KingFisher Pure Plasmid Kit 15 Chapter 4 Storage Conditions a
10. complete resuspension of the beads shake the container vigorously or vortex briefly Instructions for KingFisher Flex with 96 Deep Well Plates These instructions are intended for plasmid DNA purification from bacterial cells pelleted from 0 5 5 ml of overnight coli cultures using the KingFisher Pure Plasmid Kit Cat No 98080196 or 98080496 and the KingFisher Flex with 96 deep well plates An OD of 2 0 6 0 for cultures with high copy number plasmids ensures that bacteria have reached the proper growth density for harvesting and plasmid DNA isolation Using cultures that have OD readings gt 6 0 may lead to incomplete processing of the bacterial lysate and decreased purity of isolated plasmid DNA Thermo Scientific KingFisher Pure Plasmid Kit 17 Chapter 5 Protocols and Pipetting Instructions When using the KingFisher Pure Plasmid Kit for the first time add the RNase A solution included in the kit to the Resuspension Solution Then prepare the Wash Buffer 1 and Wash Buffer 2 For detailed 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 15 256 Do not shake the Lysis Buffer vigorously 1 Resuspend pelleted bacterial cells in 200 ul of Resuspension Solution containing RNase A The bacterial pellet should be compl
11. ep well 96 plate Add the following reagents to the rows see the table below Note that row B is reserved for the tip comb and should be left empty Note that rows C and D are also left empty Resuspend the KingFisher Magnetic Beads well e g by vortexing before removing them from the bottle Slowly aspirate 500 ul of the clear lysates and transfer them to row A Pipet carefully from the top of the supernatant to avoid touching the pelleted flocculent debris Mix the content of the Sample plate immediately by shaking the plate 4 6 times Plate name ee and type Row name Content Sample volume 5 per well Plasmid DNA A Bacterial Bacterial cell lysate 500 pl plate lysates Isopropanol a KingFisher Magnetic 250 yl leep well 96 Beads 25 ul plate B Tip 12 tip comb Empty C Empty Empty Empty D Empty Empty Empty E Wash 1_1 Wash Buffer 1 800 ul F Wash 1_2 Wash Buffer 1 700 ul G Wash 2_1 Wash Buffer 2 700 ul H Wash 2_2 Wash Buffer 2 700 ul Resuspend the KingFisher Magnetic Beads by vortexing thoroughly before use Thermo Fisher Scientific Thermo Scientific KingFisher Pure Plasmid Kit 21 Chapter 5 Protocols and Pipetting Instructions 8 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
12. ep well head elution in a KingFisher Flex 96 50 150 pl 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 DNA avoid the lowest permitted elution volumes in the KingFisher instruments The Elution Buffer should cover the KingFisher Magnetic Beads completely and any possible magnetic bead pellet s should be completely resuspended In addition the volume of Elution Buffer should be adequate for efficient mixing of the beads in order to obtain a maximal release of the purified DNA from the beads Decontamination and Disinfection of Sample Material You should decontaminate the sample material and the reagents and plastics that have been in contact with the sample material in order to minimize the risk of contamination Use a decontaminant such as Virkon paying due attention to the manufacturer s instructions You should also take care of the appropriate treatment and or disposal of waste 26 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Troubleshooting Problem Possible cause and actions Old bacterial culture Prepare new starter c
13. ep well plate 50 pcs 95040480 KingFisher Flex 24 deep well plate sterile 50 pcs 97003530 KingFisher Duo Combi pack for Microtiter deep well 1 box 96 plate tips combs plates and elution strips for 96 samples 32 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Notes Thermo Fisher Scientific Thermo Scientific KingFisher Pure Plasmid Kit 33 Notes 34 Thermo Scientific KingFisher Pure Plasmid 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
14. etely resuspended by shaking or pipetting up and down until no cell clumps remain 2 Add 200 ul of Lysis Solution and mix gently by shaking the samples 4 6 times until the solution becomes viscous and slightly clear Incubate for 2 min at room temperature NOTE Do not shake intensively to avoid shearing chromosomal DNA Do not incubate for more than 2 min to avoid denaturation of supercoiled plasmid DNA 3 Add 200 ul of Neutralization Solution and mix immediately by shaking the samples 4 6 times 4 Add 50 ul of isopropanol 100 and mix immediately by shaking the samples 4 6 times 5 To clear the lysate centrifuge the samples for 10 min at 3 800 4 000 x g to pellet the cell debris and the chromosomal DNA 6 Prepare the Sample plate i e a Thermo Scientific Microtiter deep well 96 plate as follows Add 25 ul of magnetic bead suspension and 250 ul of isopropanol to each well Slowly aspirate 500 ul of the clear lysates and transfer them to the Sample plate Pipet carefully from the top of the supernatant to avoid touching the pelleted flocculent debris Mix the content of the Sample plate immediately by shaking the plate 4 6 times Leave the plate at room temperature while the other plates are being filled 18 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Add the following reagents to the Sample plate Plate Ch
15. ingFisher Pure Plasmid Kit 9 Chapter 2 Product Description Pelleted bacterial cells are resuspended and subjected to SDS alkaline Iysis to liberate plasmid DNA The Iysate is neutralized allowing denatured plasmid DNA to reanneal Meanwhile cell debris such as proteins chromosomal DNA and SDS are precipitated and can be pelleted by centrifugation Next plasmid DNA binds to the surface of the KingFisher Magnetic Beads and impurities are effectively removed during the subsequent wash steps High quality plasmid DNA is eluted into the Elution Buffer and is ready for subsequent downstream processes Kit Specifications The KingFisher Pure Plasmid Kit is designed for rapid automated preparation of highly pure plasmid DNA from coli strains using KingFisher magnetic particle processors Fresh or frozen overnight coli culture can be used High quality plasmid DNA can be obtained from various coli strains including DH10B DH5a JM109 JM107 or TOP10 Common plasmid vectors of various length and copy number can be efficiently purified by the kit The procedure is optimized for use with bacterial cultures grown in Luria Bertani LB media The use of rich growth media may give higher yields The approximate processing time is 40 minutes for the purification of 96 samples on the KingFisher Flex and 12 samples on the KingFisher Duo The obtained DNA can be used directly in various downstream applications Typically 5 12 ug
16. iology grade e 100 isopropanol molecular biology grade Table 1 2 Thermo Scientific KingFisher magnetic particle processors Cat No Product 5400100 KingFisher Duo magnetic particle processor 5400630 KingFisher Flex magnetic particle processor with 96 deep well head 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 Plasmid 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 Plasmid Kit 7 Thermo Fisher Scientific Product Description Introduction The KingFisher Pure Plasmid Kit is designed for rapid automated purification of high copy number plasmids f
17. nd Preparation of the Reagents Table 4 1 Instructions for the preparation of the buffers Add the indicated volume per bottle 96 samples 384 samples Cat No 98080196 Cat No 98080496 Wash Buffer 1 Wash Buffer 2 Wash Buffer 1 Wash Buffer 2 Concentrated buffer 110 ml 60 ml 110 ml 60 ml Isopropanol 55 ml 55 ml Ethanol 96 100 55 ml 162 ml 55 ml 162 ml Total volume 220 ml 222 ml 220 ml 222 ml After preparing each solution mark the bottle to indicate that the step has been completed The buffers can be stored at room temperature 16 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Before beginning the DNA purification protocol carefully read through the Thermo Scientific KingFisher Flex User Manual Cat No NO7669 or the Thermo Scientific KingFisher Duo User Manual Cat No N12420 and the Thermo Scientific Binalt Software for KingFisher Instruments version 3 2 User Manual Cat No N07974 Bindlt Software 3 2 protocols for the KingFisher Pure Plasmid 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
18. of plasmid DNA can be purified from 1 ml of overnight bacterial culture with high copy number plasmid with an A A ratio of gt 1 7 2 0 The yields of acquired purified DNA depend on the bacterial strain plasmid copy number and the method of culturing 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 10 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Product Description Chapter 2 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 articles are dispensed onto the plates according to the corresponding structions Dispensing can be carried out manually or partially utomatically using automatic dispensers for example the Thermo cientific Multidrop Combi and or the Thermo Scientific VersetteTM hermo Scientific Bindlt Software 3 2 can be used for running ready made and optimized protocols for the Thermo Scientific KingFisher
19. r 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 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 Elution Buffer can be modified depending on user requirements concerning the purified DNA concentration The final DNA concentration may increase if the purified DNA is eluted into a smaller than recommended volume of Elution 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 Plasmid Kit 25 Chapter 6 General Information Table 6 1 Available elution volumes of the KingFisher Flex and KingFisher Duo o A Elution KingFisher instrument omnes KingFisher Flex with 96 de
20. r use in downstream applications NOTE The final DNA concentration in the Elution Buffer may increase if the purified plasmid DNA is eluted into a smaller than recommended volume of buffer but this can slightly reduce the overall DNA yield 22 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 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 g 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 260 300 e Total amount of DNA isolated concentration x volume of sample in ml e Purity of DNA sample Aj Az Aggy Agog e Concentration of DNA sample 50 ug ml x A X dilution factor Thermo Scientific KingFisher Pure Plasmid 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 It is strongly recommended that the volume of reagents in each well o
21. rom an overnight coli culture using Thermo Scientific KingFisher instruments The plasmid DNA purified using the KingFisher Pure Plasmid Kit is of high quality and free of proteins genomic DNA nucleases and other contaminants or inhibitors It is therefore suitable for direct use in many different downstream applications such as transformation of bacteria PCR polymerase chain reaction restriction endonuclease digestion automated sequencing in vitro transcription and other enzymatic reactions Intended Use The KingFisher Pure Plasmid Kit is developed for purification of plasmid DNA from cultured col 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 Plasmid 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 Pure Plasmid Kit for any particular use Principle and Procedure The KingFisher Pure Plasmid Kit uses magnetic particle technology for DNA purification The Thermo Scientific KingFisher technology combines the speed and efficiency of DNA purification with easy handling of magnetic particles The purification process requires no phenol chloroform extraction and needs very little hands on time Thermo Scientific K
22. ructions 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 15 256 Do not shake the Lysis Buffer vigorously 1 Resuspend pelleted bacterial cells in 200 ul of Resuspension Solution containing RNase A The bacterial pellet should be completely resuspended by shaking or pipetting up and down until no cell clumps remain 2 Add 200 ul of Lysis Solution and mix gently by shaking the samples 4 6 times until the solution becomes viscous and slightly clear Incubate for 2 min at room temperature NOTE Do not shake intensively to avoid shearing chromosomal DNA Do not incubate for more than 2 min to avoid denaturation of supercoiled plasmid DNA 20 Thermo Scientific KingFisher Pure Plasmid Kit Thermo Fisher Scientific Protocols and Pipetting Instructions Chapter 5 3 Add 200 pl of Neutralization Solution and mix immediately by shaking the samples 4 6 times 4 Add 50 ul of isopropanol 100 and mix immediately by shaking the samples 4 6 times 5 To clear the lysate centrifuge the samples for 10 min at 3 800 4 000 x g to pellet the cell debris and the chromosomal DNA 6 Take one empty Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip 7 Prepare the Plasmid DNA plate i e a Microtiter de
23. s dry as this may result in lower elution efficiency Use only Thermo Scientific plates strips and tip combs with the KingFisher instruments Use of products from other manufacturers may cause unsuitable mixing and affect the yield of purified DNA Low purity Prolonged storage of the sample material may reduce the quality and quantity of the plasmid DNA Insufficient washing causes impurities in the eluted DNA Residual salt remaining in the plasmid preparation may inhibit downstream enzymatic reactions Use the correct order for the Wash Buffers Follow the instructions to prepare the Wash Buffers on page 16 RNA contamination RNase A was not added to the Resuspension Solution Ensure that the RNase A was added to the Resuspension Solution as described on page 15 Genomic DNA contamination Samples vigorously vortexed or shook during cell lysis or neutralization steps To avoid genomic DNA contamination mix the solution by gently inverting the tube or by shaking the plate 5 8 times during the lysis and neutralization steps Do not allow the cell lysis step to proceed for more than 2 min Do not cultivate cells longer than 16 h in LB media Ensure that isopropanol was added before centrifugation Transfer lysed sample carefully from the top of the supernatant to avoid touching the pelleted flocculent Residual genomic DNA can be removed from purified plasmid DNA by treatment using T7 DNA Polymerase
24. sher instrument in use Thermo Scientific KingFisher Pure Plasmid Kit 29 Thermo Fisher Scientific Ordering Information Table B 1 KingFisher Pure Plasmid Kits Cat No Product Package size 98080196 KingFisher Pure Plasmid Kit 96 98080496 KingFisher Pure Plasmid 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 Plasmid Kit 31 Appendix B Ordering Information Table B 3 KingFisher Duo consumables Cat No Product Package size 97003500 KingFisher Duo 12 tip comb for Microtiter deep well 50 pcs 96 plate 97003510 KingFisher Duo 6 tip comb for KingFisher Flex 24 48 pcs deep well plate 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 de
25. t When the KingFisher Flex is to be run as a standalone instrument transfer the PURE_Plasmid_Flex96 protocol to the KingFisher Flex The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Binalt Software for KingFisher Instruments version 3 2 User Manual Thermo Scientific KingFisher Pure Plasmid Kit 19 Chapter 5 Protocols and Pipetting Instructions 10 After the run is completed remove the plates and store the purified DNA When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and switch off the instrument Store the purified plasmid 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 plasmid 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 These instructions are intended for DNA purification from bacterial cells pelleted from 0 5 5 ml of overnight co cultures using the KingFisher Pure Plasmid Kit Cat No 98080196 or 98080496 and the KingFisher Duo with 12 pin magnet head When using the KingFisher Pure Plasmid Kit for the first time add the RNase A solution included in the kit to the Resuspension Solution Then prepare the Wash Buffer 1 and Wash Buffer 2 For detailed inst
26. t formats Processing 0 40004 200 5000 un 30 1000 u 200 5000 pi volume Capacity Up to 96 Up to 24 Up to 12 Upto6 samples per samples per samples per samples per run sample run sample run sample run sample volume approx volume approx volume approx volume approx 200 ul 1 ml 200 ul 1 ml Magnetic 96 inter 24 format for 12 pin magnet 6 pin magnet head 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 ull plate 50 1000 ul plate 96 well PCR 200 5000 ul KingFisher Duo 200 5000 ul plate skirted elution strip 20 100 ul 80 130 yl 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
27. ulture by inoculating a freshly isolated single bacterial colony in antibiotic containing growth medium and grow bacteria according to standard protocols Incomplete bacterial cell lysis It is essential that the cell pellet is completely resuspended in Resuspension Solution prior to lysis There should be no visible cell clumps before adding the Lysis Solution Check the Lysis Solution for salt precipitation before each use Redissolve any precipitate by warming the solution to 37 C then mix well and cool to 25 C before use Use overnight culture with an OD 2 6 After centrifugation avoid transferring pelleted cell debris to a new tube or deep well plate prefilled with 25 jul of magnetic bead suspension and 250 ul of isopropanol Continued Thermo Scientific KingFisher Pure Plasmid Kit 27 Appendix A Troubleshooting Cont Possible cause and actions Low DNA yield Isopropanol and or ethanol were not added to Wash Buffer 1 Ensure that isopropanol and ethanol were added to Wash Buffer 1 before the first use Follow the instructions to prepare Wash Buffer 1 on page 16 Ethanol was not added to Wash Buffer 2 Ensure that ethanol was added to Wash Buffer 2 before the first use Follow the instructions to prepare Wash Buffer 2 on page 16 There should be an adequate volume of Elution Buffer to completely cover the KingFisher Magnetic Beads during the elution step Do not let the KingFisher Magnetic Bead
28. wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Elution Buffer 100 ul 9 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on the Plasmid DNA plate 9 Start the PURE_Plasmid_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 Connect the PC with Bindlt Software 3 2 to the KingFisher Duo Start the PURE_Plasmid_Duo protocol Insert the Plasmid 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 the PURE_Plasmid_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 7 After the run is completed remove the plate and elution strip and store the purified DNA When the protocol is completed remove the plate and elution strip according to the instructions on the KingFisher Duo display and switch off the instrument Store the purified plasmid DNA accordingly The purified DNA is ready fo
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