QuickGene DNA tissue kit S (DT-S)
Contents
1. Set the discharge tray and check the collection holder and cartridge holder settings into correct positions Press the DISCHARGE after closing 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 fromthe tissue QuickGene DNA whole blood kit S DB S Dedicated reagent kit for QuickGene series to isolate the Genomic DNA from whole blood o QuickGene RNA tissue kit S RTS Dedicated reagent kit for QuickGene series to purify the total RNA from the tissue tttstCS QuickGene RNA cultured cell kit S _RC S Dedicated reagent kit for QuickGene series to purify the total RNA from cultured cell QuickGene Plasmid kit S PLS Dedicated reagent kit for QuickGene series to extract the Plasmid DNA O O OOOO OO 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 17 18 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 fujif2. 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 QuickGene DNA tissue kit S is specifically designed for genomic DNA isolation from 5 mg of tissue sample 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 tissue kit S is evaluated routinely on a lot to lot uniformity e QuickGene DNA tissue kit S is tested for contaminations of other DNA DNase and bacteria 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 Proteinase K EDT Storage of Proteinase K EDT at 2 C to 8 C is recommended to prolog the life after open QuickGene DNA tissue kit S Tissue Lysis Buffer MDT If the precipitates are contained in Tissue Lysis Buffer incubate the bottle in a water bath at 55 C and mix with inversion the bottle intermittently until the precipitates are dissolved After dissolving the Tissue Lysis Buffer coo
3. Preparation with QuickGene series Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System 4a Select DNA TISSUE mode Press Start Button t Genomic DNA 4b Default elution volume 200 Notice 1 Tissue Lysis 1a Strictly maintain the slice of mouse tail volume at 5 mg about 5 mm but depending on the animals during the experiments Keeping the samples at room temperature for a long time degrades the genomic DNA or lowers the yield 1b When the samples are frozen thaw the samples to room temperature before using and soak them in the Tissue Lysis Buffer MDT immediately When the samples are fresh soak 5 mg of sliced tissues in the Tissue Lysis Buffer MDT immediately 1c Storage of Proteinase K EDT at 2 C to 8 C is recommended to prolog the life after open QuickGene DNA tissue kit S 1d The incubation time may change depending on the sample conditions and descriptions Dissolve the tissue completely 1e If any coat or aggregates are present in the lysate remove them by centrifugation 8 000xg or 10 000 rpm 3 min at room temperature or change the protocols by referring to troubleshooting Dissolve the samples completely by shaking and incubation 2 RNase Treatment Optional RNA can purify with Genomic DNA without RNase treatment If the contaminant RNA has negative effects on next experiments treat with RNase on this step
4. 2a Use the recommended RNase see 4 Other materials not supplied in this kit If you use the RNase Invitrogen Cat 12091 please add 60 ul for each samples 20 ul of 100 mg ml RNase Sigma Cat R5125 can digest the total RNA of 5 mg of Balb c mouse 7 weeks old tail completely 2b Mix well the RNase and lysate 2c Flash spin down completely 3 Addition of Reagents 3a Mix completely the Lysis Buffer LDT 180 ul and gt 99 ethanol 240 ul before using 3b Mix completely the Lysis buffer LDT and sample solution If the mixing is not enough by Vortex mixer use the pipetting tapping inverting etc 3c If the solution become clouded after adding gt 99 ethanol due to low room temperature incubate the sample solution at 55 C Cool down the samples to room temperature before next step 3d Transfer the whole lysate into the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Perform isolation within 30 min after lysate preparation 4 Preparation with QuickGene series 4a Transfer the whole lysate into the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Perform isolation within 30 min after lysate preparation 4b Standard 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 13 14 8 3 Genomic DNA isolation using the QuickGene series Automatic Nucleic
5. 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 Please take Discharge before isolation with QuickGene series Automatic Nucleic Acid Isolation System every time 1 Selection of isolation mode Select DNA TISSUE mode for genomic DNA isolation 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 regents of Wash Buffer WDT with gt 99 ethanol and Elution Buffer CDT 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 contamination e Read t
6. 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 fuhsing mail hungchong com tw URL http www FUJIFILM COM TW Appendix 1 DNA TISSUE mode is set in the following parameter DNA TISSUE 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 90
7. W FUJIFILM HANDBOOK QuickGene DNA tissue kit S DT S For Isolation of Genomic DNA from tissue samples 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 9 Troubleshooting 10 Ordering Information 11 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 tissue kit S is used with the QuickGene series Automatic Nucleic Acid Isolation System high quality and high yield genomic DNA can be isolated and a
8. der Add the reagents to micro tubes in the following order when preparing the lysate from tissues Tissue lysate gt Lysis Buffer LDT gt 99 ethanol or mouse tail lysate gt Lysis Buffer LDT with gt 99 ethanol mixture Too much tissues Reduce the amount of tissues below the specified amount Insufficient homogenization following the addition of Lysis Buffer LDT Required volume of ethanol was not added to Wash Buffer WDT Vortex sufficiently 15 sec immediately after Lysis Buffer LDT addition Always confirm that the required volume of ethanol is added to the Wash Buffer WDT prior to use Old Wash Buffer WDT including ethanol was used 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 Insufficient amounts of reagents used If aggregates are present in the lysate apply them along with the lysate to the cartridge Make sure that sufficient amount of reagent are in the reagent bottles Supplying the precipitates in reagents See 4 section 2 Clogging the cartridge Cause Possible Solution Excess amount of tissues were used Reduce the amount of tissues below the specified amount Insufficient homogenization following the addition of Lysis Buffer LDT Vortex sufficiently 15 sec immediately after Lysis Buffer LDT addition Incom
9. e life after open QuickGene DNA tissue kit S 1d The incubation time may change depending on sample conditions and descriptions Dissolve the tissue completely 1e If any aggregates are present in the lysate remove them by centrifugation 8 000xg or 10 000 rpm 3 min at room temperature or change the protocols by referring to troubleshooting Dissolve the samples completely with shaking and incubation 2 RNase Treatment Optional RNA would purify with Genomic DNA without RNase treatment If the contaminant RNA has negative effects on next experiments treat with RNase on this step 2a Use the recommended RNase see 4 Other materials not supplied in this kit If you use the RNase Invitrogen Cat 12091 add 60 ul for each sample 20 ul of 100mg m RNase can digest the total RNA of 5 mg of liver tissue completely 2b Mix well the RNase and lysate 2c Flash spin down completely 3 Addition of Lysis Buffer 3a Mix completely with the Lysis Buffer LDT and sample solution If the mixing is not enough by Vortex mixer use the pipetting tapping inverting etc 3b amp 3d Incubate the sample solution at 70 C if the solution becomes clouded at LDT addition step 4 Addition of Ethanol 4a Use gt 99 Ethanol 4b Mix completely the sample solution and gt 99 ethanol If the mixing is not enough by Vortex mixer use the pipetting tapping inverting etc 4c If the solution becomes clouded after adding gt 99 ethanol due to
10. e tissue completely 1d Flash spin down 1e Transfer the supernatant to new 1 5 ml micro tube 2 RNase Treatment Optional Add RNase A 2041 2a Tap the tube to mix the solution 2b Flash spin down 2c Set it down at room temperature for 2 min 3 Addition of Lysis Buffer _ Add LDT 1801 Mix thoroughly by vortexing for 15 sec 3a Flash spin down Incubate at 70 C for 10 min 3b Flash spin down 4 Addition of Ethanol Add gt 99 Ethanol 240 UI 4a Mix thoroughly by vortexing for 15 sec 4b Flash spin down 4c Lysate 4d 5 Preparation with QuickGene series Transfer the whole lysate to the cartridge of QuickGene series Automatic Nucleic Acid Isolation System 5a Select DNA TISSUE mode Press Start Button Genomic DNA 5b Default elution volume 200 Notice 1 Tissue Lysis 1a Strictly maintain the sample volume at 5 mg during the experiments It is ideal to cut the tissue into small pieces for quick dissolution Keeping the samples at room temperature for a long time degrades the genomic DNA or lowers the yield 1b When the samples are frozen thaw the samples to room temperature before using and soak them in the Tssue Lysis Buffer MDT immediately When the samples are fresh soak 5 mg of sliced tissues in the Tssue Lysis Buffer MDT immediately 1c Storage of Proteinase K EDT at 2 C to 8 C is recommended to prolog th
11. from the collection tube holder e As genomic DNA is eluted from the Cartridge s CA using 200 ul of Elution Buffer CDT the volume of recovered total 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 15 9 Troubleshooting Review the information below to troubleshoot the experiments with QuickGene DNA tissue kit S For system related problems e g when an error message appears see the QuickGene series 16 user s manual 1 Low yield or no DNA obtained Cause Possible Solution Storage condition of sample Incorrect storage condition induces low yield Try different storage conditions that may vary with sample volume and storage period Incompletely dissolved samples Soak the 5 mg of sliced tissues in the Tissue Lysis Buffer MDT with ProteinaseK immediately Cut the tissue into small pieces Extend the incubation time in the lysis step Dissolve the tissue completely with shaking and incubation Prepare the sample volume to 200 u before LDT addition Reagents and tissues added in the wrong or
12. he user s manual for the QuickGene series Automatic Nucleic Acid Isolation System for details for setting reagents The standard default elution volume is 200 ul but you may change the setting of elution volume less than default volume minimum 50 UI In case of setting to 50 ul yield may decline 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 preparation 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 WDT with gt 99 ethanol Collection Buffer CDT 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
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14. l down the bottle to room temperature before using Lysis Buffer LDT 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 WDT 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 WDT with gt 99 ethanol and Elution Buffer CDT Prepare the requirements of Wash Buffer WDT with gt 99 ethanol and Elution Buffer CDT 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 Table3 Buffer volume and the number of samples to set in the QuickGene System Number of samples WDT with Ethanol CDT 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 30 ml 8 2 Sample preparations e Basically the QuickGene DNA tissue kit S is specifically designed for genomic DNA isolation from 5 mg of tissue sample H
15. low room temperature incubate the sample solution at 55 C Cool down the samples to room temperature before next step 4d Transfer the whole lysate into the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Perform isolation within 30 min after lysate preparation 5 Preparation with QuickGene series 5a Transfer the whole lysate into the cartridge of QuickGene series Automatic Nucleic Acid Isolation System Perform isolation within 30 min after lysate preparation 5b Standard 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 11 12 lt Preparation workflow from the slice of mouse tail gt 1 Tissue Lysis Empty 2 ml Micro centrifuge Add slice of mouse tail 5 mg 1a Add MDT 180H 1b lt Add EDT 204l 1c Incubate for over night on Rotary Shaker at 55 C and dissolve the tissue completely 1d Flash spin down 1e Transfer the supernatant to new 1 5 ml micro tube 2 RNase Treatment Optional j lt Add RNase A 20 2a Tap the tube to mix the solution 2b Flash spin down 2c Set it down at room temperature for 2 min 3 Addition of Reagents Add pre mixed LDT 180 Ul with gt 99 Ethanol 240 u1 420 UI 3a Mix thoroughly by vortexing for 15 sec 3b Flash spin down 3c t Lysate 3d 4
16. lso purified from tissue samples In addition DNA from 8 sets of tissue lysate samples can be simultaneously extracted in only 13 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 Proteinase K EDT Tissue Lysis buffer MDT Lysis buffer LDT Wash buffer WDT Elution buffer CDT Cartridges CA Collection tubes CT Caps CAP Waste tubes WT 3 Storage conditions Store all reagents at 15 C to 28 C Storage of Proteinase K EDT at 2 C to 8 C is recommended to prolog the life after open QuickGene DNA tissue kit S 4 Other required materials not supplied in this kit Reagents e gt 99 Ethanol e RNase A Optional Instruments and equipments e QuickGene series Automatic Nucleic Acid Isolation System e 2 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 e Rotary shaker with heater Table1 Recommended centrifuge tubes Size of QuickGene series centrifuge tube holder Type of centrifuge tube Product name Examples Standard Large centrifuge tube for WDT Small cent
17. ommendations http www fujifilm co jp msds Proteinase K EDT 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 Tissue Lysis Buffer MDT 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 LDT 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 WDT 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 Elution Buffer CDT 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 Handle the Lysis buffer LDT in a well ventilated area and keep away from heat sparks and flame Keep container tightly closed It might be harmful to inhale 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
18. owever it would be able to isolate the genomic DNA from 20 mg of tail of 6 weeks old female Balb c mice e The yield and preparation time may change with sample volume variety of tissues tissue storage condition and lysate condition e 20 mg of Liver of 6 weeks old female Balb c mice would be able to isolate the genomic DNA without RNase treatment Though the cartridge on QuickGene series may clog or elution time may extend When using more than 5 mg of Liver sample RNA digestion may incomplete Pretest the RNase treatment condition in this case e 10 mg of Lung or Kidney of 6 weeks old female Balb c mice would be able to isolate the genomic DNA without RNase treatment Though the cartridge on QuickGene series may clog or elution time may extend e Soak the 5 mg of sliced tissue in the Tissue Lysis Buffer MDT immediately Freeze the lysate with liquid nitrogen and store at 20 C or 80 C if you do not prepare the samples immediately e Keeping the samples at room temperature for a long time and or repeatedly freezing or thawing degrades the genomic DNA or lowers the yield e Accurately measure the buffer volume during the experiments lt Preparation workflow from the mammalian tissue samples gt 1 Tissue Lysis Empty 2 ml Micro centrifuge lt Add slice of mammalians tissue 5 mg 1a lt Add MDT 1801 1b lt Add EDT 204l 1c Incubate for over night on Rotary Shaker at 55 C and dissolve th
19. plete dissolved samples See 1 section Clogging the cartridge with insoluble Remove the insoluble with centrifuge before Lysis Buffer LDT is added 3 Subsequent experiments e g PCR unsuccessful Cause Possible Solution Improper amount of DNA used for subsequent experiments Determine the concentration based on the absorbance at 260 nm Genomic DNA would degrade Soak the 5 mg of sliced tissues in the Tissue Lysis Buffer MDT immediately or freeze the lysate with liquid nitrogen and store at 20 C or 80 C if you do not prepare the samples immediately 4 Supplying the precipitates in reagents Cause Possible Solution Stored at low temperature Store at 15 C to 28 C If the precipitates are contained incubate the bottle of MDT in a water bath at 55 C and bottle of LDT at 37 C and mix with inversion the bottle intermittently until the precipitates are dissolved 5 Solutions become clouded after adding gt 99 ethanol in preparation workflow from the mammalians tissue or LDT from the slice of mouse tail with adding gt 99 ethanol mixture in preparation workflow Cause Possible Solution Low temperature Incubate the sample solution at 55 C to dissolve and cool down the samples to room temperature before next step 6 The collection tubes are empty after the elution Cause Possible Solution Missed the discharge
20. rifuge tube for CDT BD Falcon 50 ml conical tube BD Falcon 15 ml conical tube Large Large centrifuge tube for WDT Small centrifuge tube for CDT BD Falcon 175 ml conical tube 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 WDT with ethanol and Elution buffer CDT Table2 Recommended Rnase A for optional process Product Name Manufacture Cat No Preparation Ribonuclease A Sigma R5125 1 2 Ribonuclease A Sigma R5500 1 2 Ribonuclease A Sigma R6513 1 Ribonuclease A Sigma R4642 Ribonuclease A MP Biomedicals 101076 1 2 RNase A AMRESCO 0675 1 2 RNase A QIAGEN 19101 RNase A Invitrogen 12091 Preparation 1 Prepare 100 mg ml solution with 10 mM Tris HCI pH 7 5 and 15 mM NaCl 2 Incubate at 100 C for 15 min to deactivate DNase 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 rec
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