QuickGene DNA whole blood kit S (DB-S)
Contents
1. The dissolved protease EDB will be able to store for two months at 4 C The enzyme will be stable for a longer period at 20 C Recommend to avoid repeated freezing and thawing Lysis Buffer LDB Mix thoroughly before using If the precipitates are contained in Lysis Buffer incubate the bottle in a water bath at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved After dissolving the Lysis Buffer cool down the bottle to room temperature before using Wash Buffer WDB Provide the concentrated solution Add 160 ml of gt 99 ethanol into the bottle and mix with inversion the bottle gently at the beginning of use Requirements of Wash Buffer WDB with gt 99 ethanol and Elution Buffer CDB Prepare the requirements of Wash Buffer WDB with gt 99 ethanol and Elution Buffer CDB according to the number of samples for isolation refer to the following table Take some of the buffers into each tube and set the tubes in the QuickGene series system tube holder See the user s manual of QuickGene series Automatic Nucleic Acid Isolation System Table2 Buffer volume and the number of samples to set in the QuickGene System Number of samples WDB with Ethanol CDB 8 26 ml 8 ml 16 44 ml 11 ml 24 62 ml 13 ml 32 80 ml 15 ml 40 99 ml 17 ml 48 117 ml 19 ml 56 135 ml 21 ml 64 154 ml 22 ml 72 172 ml 24 ml 80 190 ml 26 ml 88 209 ml 28 ml 96 227 ml 32. A Tel 1 203 324 2000 ext 6112 1 800 431 1850 ext 6112 in the U S Fax 1 203 351 4713 E mail SSG fujimed com URL http lifescience fujifilm com lt Europe excl UK and Ireland gt Fuji Photo Film Europe GmbH Heesenstr 31 40549 Dusseldorf Germany Tel 49 211 5089 174 Fax 49 21 1 5089 139 E mail lifescience fujifilmeurope de URL http www fujifilm de lt UK Ireland gt Fuji Photo Film U K Unit 12 St Martinsfs way St Martin s Business centre Bedford MK42 QLF UK Tel 44 1234 245291 Fax 44 1234 245293 E mail lifesciences fuji co uk URL http lifescience fujifilm com lt China gt Fuji Photo Film China Investment Co Ltd 31st floor Hong Kong New World Tower No 300 Huai Hai Zhong Road P R China Tel 86 21 3302 4655 Fax 86 21 6384 7700 E mail wgxiang fujifilm com cn URL http www fujifilm com cn Distributors lt Australia New Zealand gt Berthold AUSTRALIA PTY Ltd 40 Clements Ave Bundoora Vic 3083 Australia Tel 61 3 9467 6277 Fax 61 3 9467 7493 E mail rafael berthold com au URL http berthold com au lt Korea gt Shinki Hi Tec GUNWHA Bldg 7 1 Yangjae 1 dong Secho gu Saoul Korea 113 887 Tel 82 2 572 1600 Fax 82 2 572 0058 E mail info skhitec co kr URL http www skhitec co kr lt Taiwan gt HUNG CHONG CORP No 38 Sec 6 Min Chuan E Road Taipei Taiwan Tel 886 2 2791 1188 Fax 886 2 2794 2248 E mail fuhsi
3. the 30 ul of EDB to bottom of 2 ml micro tube 1b Add 200 LI of whole blood into the micro tube and then add 250 ul of LDB immediately Leaving the samples long time before addition of LDB may be reduced the yield 1c Mix whole blood sample and LDB by pipetting 5 times It is very important to mix thoroughly the sample with pipetting after addition of LDB 2 Vortexing for 15 sec with maximum speed and flash spin down to recover the lysate from cap and tube wall Recommending vortex speed is 2 500 rpm and more If you do not have such a vortex mixer pipetting completely at 1c Incomplete mixing at this time the sample will be clogged the cartridge of QuickGeme series Automatic Nucleic Acid Isolation System or low yield 3 Incubate at 56 C 2 min The maximum incubation time is 5 min 4 Add 250 ul gt 99 Ethanol and vortexing for 15 sec with maximum speed Flash spin down to recover the lysate from cap and tube wall 5 Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Perform isolation within 30 min after lysate preparation If aggregates are present in the lysate apply them along with the lysate to the cartridge 6 Default elution volume is 200 ul but you may change the setting of elution volume less than default volume minimum 50 ul In case of setting to 50 ul yield may decline The standard yield of eluted genomic DNA is 4 8 ug from 200 ul whole blood Store the elut
4. 0 ml 8 2 Sample preparations e The QuickGene DNA whole blood kit S is specifically designed for genomic DNA isolation from 200 ul of whole blood e Recommend to use the whole blood collected in EDTA 2Na EDTA 2K or heparin e The yield will depend on the sample condition e Use the kit at room temperature 15 C to 30 C When using the kit at lower or higher temperatures the expected yield may not be obtained e Accurately measure the buffer volume during the experiments lt Preparation workflow from whole blood gt 6 Empty 2 ml Micro centrifuge tube Add EDB solution 3041 1a Add whole blood 200 ul 1b Add LDB 25041 1c Pipetting 5 times Mix thoroughly by vortexing maximum speed or gt 2 500 rpm for 15 sec Flash spin down Incubate at 56 C 2 min lt Add gt 99 Ethanol 250 ul Mix thoroughly by vortexing maximum speed or gt 2 500 rpm for 15 sec Flash spin down Y Lysate Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Select DNA WHOLE BLOOD mode Press START Button Genomic DNA Default elution volume 200 Notice 1 Follow the protocol of 1a to 1c exactly If you change the protocol may be reduced the yield 1a Add 3 3 ml of nuclease free ultra pure water to the vial containing the freeze dried protease and dissolve it carefully Put
5. DNA solution will be 200 ul Cover with the Caps CAP on the Collection Tubes CT containing the isolated genomic DNA 8 Clean up Remove the Waste Tubes WT and dispose the waste fluid according to applicable regulations Remove the cartridge holder and dispose the Cartridges CA Warning Disposal of waste fluid and consumables When using the potentially infectious samples for experiments dispose them according to applicable regulations 11 12 9 Troubleshooting Review the information below to troubleshoot the experiments with QuickGene DNA whole blood kit S For system related problems e g when an error message appears see the QuickGene series user s manual 1 Low yield or no DNA obtained Cause Possible Solution Reagents and whole blood added in the wrong order Excess amount of sample was used Add the reagents and samples to microtubes in the following order when preparing the lysate Protease EDB dissolved in 3 3 ml of nuclease free water gt whole blood Lysis Buffer LDB Reduce the amount of whole blood to below the specified amount Insufficient homogenization following the addition of Lysis Buffer LDB Vortex sufficiently 15 sec immediately after Lysis Buffer LDB addition Requirement volume of ethanol was not added to Wash Buffer WDB Always confirm that the required volume of ethanol was added to the Wash Buffer WDB prior to use Old Wash Buf
6. W FUJIFILM HANDBOOK QuickGene DNA whole blood kit S DB S For Isolation of Genomic DNA from whole blood Contents Introduction Components of the kit Storage conditions Other required materials not supplied in this kit Safety warnings Precautions Quality controls Protocols 8 1 Preparation of reagents 8 2 Sample preparations 8 3 Genomic DNA isolation using the QuickGene series Automatic Nucleic Acid Isolation System Troubleshooting Ordering Information Contact Information Appendix 1 1 2 3 4 5 6 7 8 Warning For research use only Not recommended and intended for diagnostic or clinical application for human and animals 1 Introduction Fuji Photo Film Co LTD developed and patented an evolutionary porous membrane to immobilize nucleic acid Because of its large specific surface area and uniform amp fine porousness QuickGene successfully isolates genomic DNA with high yield moreover with its patented thin membrane it eliminates most contaminants QuickGene also uses pressured filtration technology which cannot be successfully utilized with typical glass membranes by using pressured filtration technology new compact and automatic instruments for rapid nucleic acid purification can be produced successfully When QuickGene DNA whole blood kit S is used with the QuickGene series Automatic Nucleic Acid Isolation System high quality and high yield genomic DNA can be isolated a
7. ately with water Elution Buffer CDB Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water e Keep away the Lysis Buffer LDB from heat Do not mix with disinfectants such as bleach e For disposal of waste fluid and consumables When using potentially infectious samples for experiments dispose them according to applicable regulations 6 Precautions e Refer to the MSDS Material Safety Data Sheet for specific recommendations on properties and handling The MSDS can be obtained from the World Wide Website http lifescience fujifilm com e Refer to the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System before using 7 Quality controls e The stability of the reagents is guaranteed for one year after purchase if stored at the specified temperature 15 C to 28 C e As part of the stringent of quality assurance program in Fuji Photo Film Co LTD the performance of QuickGene DNA whole blood kit S is evaluated routinely on a lot to lot uniformity e Quality and yield of isolated genomic DNAs are checked by measuring the absorbance at 260 nm ratio of absorbance 260 nm 280 nm and PCR amplification 8 Protocols 8 1 Preparation of reagents Protease EDB Add 3 3 ml of nuclease free ultra pure water to the vial containing the freeze dried protease and dissolve it carefully
8. ed genomic DNA at 20 C for long storage 10 8 3 Genomic DNA isolation using the QuickGene series Automatic Nucleic Acid Isolation System Notice System set up and basic operations Please read the user s manual of QuickGene series Automatic Nucleic Acid Isolation System circumstantially for the details before using the system 1 Selection of isolation mode Select DNA WHOLE BLOOD mode for genomic DNA isolation from whole blood with the kit See Appendix 1 2 Setting of cartridges and tubes Open the front cover of the instrument and set the collection and waste tubes in the collection tube holder e Use the specified Collection Tubes CT and Waste Tubes WT including the kit Attach the cartridge holder to the instrument and set 1 8 cartridges in the cartridge holder e Use the specified Cartridges CA Notice Refer to the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System for details of setting cartridges and tubes Incorrect cartridge placement may result in the solution spilling or improper isolation Wear gloves during the experiments to avoid nuclease contamination 3 Setting of reagents Prepare the required volume see 8 1 Preparation of reagents of Wash Buffer WDB with gt 99 ethanol and Elution Buffer CDB into the tubes set them to the holder and put the holder to the designated positions of instrument Notice Wear gloves during the handling of reagents to avoid nuclease co
9. fer including ethanol used WDB Flash remaining Wash Buffer WRC including ethanol which may be one day old or more in the instrument prior to use Lysate is not fully applied to Cartridge s CA If aggregates are present in the lysate apply them along with the lysate to the cartridge Insufficient amounts of reagents used Make sure that sufficient amount of reagent are in the reagent bottles 2 Clogging the cartridge Cause Possible Solution Excess amount of sample was used Reduce the amount of whole blood to below the specified amount Insufficient homogenization following the addition of Lysis Buffer LDB Vortex sufficiently 15 sec immediately after Lysis Buffer LDB is added 3 Subsequent experiments e g PCR unsuccessful Cause Possible Solution Improper amount of DNA used for subsequent experiments 4 Supplying the precipitates Determine the concentration based on the absorbance at 260 nm in reagents Cause Possible Solution Stored at low temperature Store solutions at 15 C to 28 C If the precipitates are contained incubate the bottle in a water bath at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved 5 The collection tubes are empty after the elution Cause Possible Solution Missed the discharge Set the discharge tray and check the collection holder a
10. ml conical tube large Large centrifuge tube for WDB BD Falcon 225 ml conical tube BD Falcon 50 ml conical tube Centrifuge tubes are used with the QuickGene series Automatic Nucleic Acid Isolation System as containers for the wash buffer WDB with ethanol and collection buffer CDB 5 Safety warnings Warning For research use only Not recommended and intended for diagnostic or clinical application for human and animals e All reagents and items should be considered chemically and biologically hazardous Wearing a laboratory coat gloves and safety glasses during the experiments are highly recommended In case of contact between the reagents and the eyes skin or clothing wash immediately with water See the Material Safety Data Sheet for specific recommendations http www fujifilm co jp msds Protease EDB Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water Lysis Buffer LDB Poisonous if swallowed Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immediately with water Wear laboratory coat gloves and safety glasses during experiments Wash Buffer WDB Don t put reagents in eyes and be careful of accidental ingestion In case of contact between the reagents and eyes skin or clothing wash immedi
11. nd also purified from whole blood In addition DNA from 8 sets of tissue lysate samples can be simultaneously extracted in only 6 minutes The purified high quality genomic DNA is suitable for PCR restriction enzyme digestion southern blotting and other applications Please be sure to read this handbook carefully before using the kit 2 Components of the kit The kit includes the reagents necessary for 96 sets of genomic DNA isolation Protease EDB Lysis buffer LDB Wash buffer WDB Elution buffer CDB Cartridges CA Collection tubes CT Caps CAP Waste tubes WT 3 Storage conditions Store all reagents at 15 C to 28 C 4 Other required materials not supplied in this kit Reagents e gt 99 Ethanol e Nuclease free ultra pure water for dissolving proteases Instruments and equipments e QuickGene series Automatic Nucleic Acid Isolation System e 1 5 ml Micro centrifuge tubes e Centrifuge tubes see Table1 e Micropipettes and tips e Vortex mixer e Micro centrifuge c a 5 000xg e Tube stands Table1 Recommended centrifuge tubes Size of QuickGene series centrifuge tube holder Type of centrifuge tube Product name Examples Large centrifuge tube for WDB BD Falcon 50 ml conical tube Small centrifuge tube for CDB Standard Small centrifuge tube for CDB BD Falcon 15 ml conical tube r BD Falcon 175
12. nd cartridge holder setting up into correct positions Press the DISCHARGE after closed the front cover of the instrument See the QuickGene series user s manual 10 Ordering Information Product Cat QuickGene series Automatic Nucleic Acid Isolation Systems QuickGene DNA tissue kit S DT S Dedicated reagent kit for QuickGene series to isolate the Genomic DNA from the tissue QuickGene DNA whole blood kit S DB S Dedicated reagent kit for QuickGene series to isolate the Genomic DNA from whole blood QuickGene RNA tissue kit S RT S Dedicated reagent kit for QuickGene series to purify the total RNA from the tissue QuickGene RNA cultured cell kit S RC S Dedicated reagent kit for QuickGene series to purify the total RNA from cultured cel SS QuickGene Plasmid kit S PL S mr a eee one Trade Mark Falcon Becton Dickinson and Company The Polymerase Chain reaction PCR is covered by patents owned by Roche Molecular Systems and F Hoffmann La Roche Ltd 13 14 11 Contact Information http Nifescience fujifilm com Fuji Photo Film Co Ltd LIFE SCIENCE PHOTO IMAGING amp INFORMATION PRODUCTS DIVISION 26 30 Nishiazabu 2 Chome Minato ku TOKYO 106 8620 JAPAN Tel 81 3 3406 2201 Fax 81 3 3406 2158 E mail sginfo tokyo fujifilm co jp Subsidiaries lt United States Canada Mexico gt Fujifilm Medical System U S A Inc 419 West Avenue Stamford CT 06902 U S
13. ng mail hungchong com tw URL http www FUJIFILM COM TW Appendix 1 DNA WHOLE BLOOD mode is set in the following parameter DNA WHOLE BLOOD PARAMETER SET VALUE BIND PEAK 120 WASH COUNT 3 WASH PEAK 110 WASH VOL1 750 WASH VOL2 750 WASH VOL3 750 WASH VOL4 750 WASH VOL5 750 WASH DIP TM 0 WAS2 WAIT T 0 WAS2 COUNT 0 WAS2 PEAK 110 WAS2 VOL1 750 WAS2 VOL2 750 WAS2 VOL3 750 WAS2 VOL4 750 WAS2 VOL5 750 ELUT VOL 200 ELUT PEAK 100 ELUT DIP TM 0
14. ntamination e Read the user s manual for the QuickGene series Automatic Nucleic Acid Isolation System for details for setting reagents 4 Discharge Set the discharge tray and check the collection holder and cartridge holder setting for the correct positions Press the DISCHARGE after closed the front cover of the instrument Notice Because of air in the lines incorrect volume of reagents may occur without discharge operation 5 Applying the prepared samples Apply all contents of prepared lysate samples see 8 2 Sample preparations into the each Cartridge CA by using micropipettes any aggregates in the lysate should be transfered into the cartridge 6 Isolation Check if the materials Wash Buffer WDB with gt 99 ethanol Elution Buffer CDB Cartridges CA including samples Waste Tubes WT and Collection Tubes CT are well set Close the front cover of the instrument Confirm the appropriate mode on the operation panel and press the START button 7 Collection of genomic DNA After completing the process each sample result is indicated on the operation panel as follow v Check Completed normally Hyphen Not completed normally __ Underscore No cartridge or no sample Open the front cover and remove the Collection Tube s CT from the collection tube holder e As genomic DNA is eluted from the Cartridge s CA using 200 ul of Elution Buffer CDB the volume of recovered total
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