PrepSEQ® Residual DNA Sample Preparation Kit User Guide (PN
Contents
1. Poor extraction efficiency low yields Ethanol is in the Wash Solution step h on page 15 Thoroughly air dry the magnetic particles pellet in the magnetic stand for 5 minutes at room temperature Magnetic particles are attached too tightly to the tube wall during the elution step a on page 15 Place the tube in the microcentrifuge with the magnetic particles pellet oriented toward the center Spin the tube for 30 seconds to detach the magnetic particles from the tube wall into the Elution Buffer Magnetic particles are difficult to resuspend during the elution step b on page 15 Incubate the pellets at 70 C for 7 minutes Vortex the tubes three times during incubation to help resuspension Formation of precipitate in magnetic particles page 16 Incubate magnetic particles at 37 C for 10 minutes then vortex the magnetic particles at setting 7 for 30 seconds to completely resuspend the particles Particles no longer produce consistent results fine brown sandy particles and brown color in the supernatant Samples have low pH step e on page 14 Measure the pH of the sample and adjust the pH to between 6 and 8 Magnetic particles were stored at 20 C Kit contents and storage Magnetic Particles on page 7 Order new materials and store them at 4 C PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 19 D 0 me GH rm O LD e 3 2 0 ay2. 0 e e EX m o 2 PrepSEQ Residual DNA Sample Preparation Kit Protocol Troubleshooting 20 PrepSEQ Residual DNA Sample Preparation Kit User Guide APPENDIX Safety Appendix This appendix covers Chemical safety cse cete cete cete Hx ewe sehen E es det E HET 21 Chemical w stesafety i cT sue a weba Eve br e pP Pub Wek ek b n IET DR k 22 Biological hazard safety ss seks ka ku l tinet iuir tanid init iras 24 Chemical alerts Wk ccc KK KK KK KK KK KK KK KK KK KK KK KK kk kK hrs 24 Chemical safety Chemical hazard WARNING CHEMICAL HAZARD Before handling any chemicals refer to i the Safety Data Sheet SDS provided by the manufacturer and observe all warning ty P y relevant precautions N WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument A WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles N WARNING CHEMICAL STORAGE HA
3. Step 6 Without disturbing the magnetic beads remove and discard the supernatant using a Pipetman or by aspiration Step 7 Use a P200 pipettor to remove residual solution Step 8 Leave the tube lids open for 5 min to air dry see the corresponding IMPORTANT on page 15 Y Elute DNA Step 1 Add 50 uL of Elution Buffer to each tube see the corresponding IMPORTANT on page 15 Y Step 2 Vortex for 10 sec at high speed then incubate the tubes at 70 C for 7 min Vortex two or three times to resuspend particles y Step 3 Spin for 15 sec and place the tubes into the magnetic stand for 2 min Then transfer eluate to a nonstick 1 5 mL tube 12 pe Step 4 Spin for 3 min at top speed Place the tubes into a magnetic stand Y Step 5 Transfer the eluate to a nonstick 1 5 mL tube Avoid the magnetic beads see the corresponding Note on page 15 Y When done set up PCR using 10 uL of eluate See the resDNASEQ Quantitative DNA Kits Protocol Part no 4415260 for more information PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Extract Host Cell DNA Workflow for automated E coli and Vero DNA extraction The automated extraction workflow is shown below For details go to page 16 Prepare the plates Prepare the Lysis Wash 1 Wash 2 Elution and Comb loading plates according to the table on page 16 Select the instrument proto
4. 20 C 4404627 IMPORTANT White precipitate occasionally forms in the magnetic particles tube Before each use incubate the tube containing the magnetic particles at 37 C for 10 minutes then vortex the tube at setting 7 to completely resuspend the particles Automation MagMAX Express 96 DW instrument Part no 4456933 accessories include instrument plastics and accessories Materials not included in the kit Item Source or part number MagMAX Express 96 DW plate 4388476 MagMAX Express 96 DW well tip combs 4388487 MagMAX Express 96 DW magnetic head 4388435 MagMAX Express 96 DW standard plates 4388475 Magnetic Stand 96 AM10027 Vortex Adapter 60 for use with the Vortex Genie AM10014 Materials that are required for use of but are not included in the PrepSEQ Residual DNA Sample Preparation Kit include Item Source or part number Equipment Block heater for use with 2 mL tubes Manual DNA extraction involves two incubations at different settings so two heaters may be Major laboratory supplier MLS convenient Magnetic stand 16 position 4457858 Benchtop microcentrifuge for MLS 1 5 mL and 2 mL tubes Vortex Genie 2T Mixer VWR 14216 188 VWR 14216 186 Vortex Adapter 60 for use with the Vortex Genie AM10014 Consumables Disposable gloves MLS Aerosol resistant micropipette tips MLS PrepSEQ Residual DNA Sample Preparation
5. Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Required materials and preparation Item Source or part number Pipetman Pipettors P1000 P200 P20 and P10 MLS e Positive displacement e Air displacement e Multichannel IMPORTANT Do not use denatured ethanol It contains components that are not compatible with the protocol Pipettes MLS 3 Microcentrifuge tubes nonstick RNase free AM12450 p 1 5 mL 1 box 250 tubes box 2 Safe Lock PCR clean microcentrifuge tubes VWR 62111 754 e round bottom 2 mL E Reagents U Ethanol 95 MLS S Ej Isopropanol 100 MLS 5 M NaCl and 1 N NaOH solutions MLS Hydrochloric acid HCI MLS PBS solution 1X pH 7 4 free of Mg and Ca if Ambion AM9624 needed to dilute samples Safety Hazards For all chemicals read the Safety Data Sheet SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Reagent Before you use the PrepSEQ Residual DNA Sample Preparation Kit prepare the preparation following solutions PrepSEQ Binding Solution 1 Add 30 mL of 100 isopropanol to the Binding Solution bottle 2 Label the bottle to indicate that it contains isopropanol then store the bottle at ambient temperature PrepSEQ Wash Buffer Concentrate 1 Add 74 mL of 95 ethanol to the bottle that is labeled PrepSEQ Wash Solution Concentrate then mix completely 2 Label
6. 00 uL wash buffer Wash 1 plate with 300 uL wash buffer PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Automated protocol for residual DNA extraction e Lysis plate 4 Prepare samples for digestion a Add 100 uL of sample to a well of the 96 deep well plate If necessary adjust pH level using 10 N NaOH or 10 N HCI optimum range is 6 to 8 then measure to confirm the pH level If necessary centrifuge the plate at 1000 rpm for 1 minute in a regular bench top microfuge to spin down the solution on the wall Optional Add 10 uL of 5 M NaCl if the salt concentration of the sample is lower than 0 5 M Note Test samples from the early purification process often contain levels of DNA that are above the highest point of the residual DNA assay standard curve See Dilute DNA samples if necessary on page 18 Alternatively make dilutions of the extracted DNA before running the PCR reaction b Make a master mix of Proteinase K buffer and Proteinase K then add 70 uL to each sample Briefly vortex then briefly spin the master mix Incubate the mix at 56 C for 30 minutes C Place the plate in the processor then press START to begin the lysis process The instrument mixes the samples for 10 seconds at fast speed then incubates the samples at 57 C for 30 minutes mixing at slow speed Proceed to step 5a while the samples incubate 5 Prepare the lysis and bind t
7. L of freshly made Lysis Solution Mix per 100 uL of starting sample see Reagent preparation on page 9 2 Bind the DNA For all chemicals avoid contact with skin eyes and or clothing Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves a Add 30 uL of the Magnetic Particles to the 100 or 200 uL sample using a wide bore pipette tip Add 300 uL of the Binding Solution per 100 uL of starting sample close the caps and invert the tubes twice to mix then vortex the tubes for 5 minutes at setting 7 Quick spin the tubes in a microcentrifuge for 15 seconds to pellet the magnetic particles then place the tubes in the magnetic stand and let the tubes stand for 5 minutes or until the solution is clear Without disturbing the magnetic beads remove the supernatant using a Pipetman or by aspiration 3 Wash the DNA 14 PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Manual protocol for residual DNA extraction a Remove the tubes from the magnetic stand then add 300 uL of Wash Solution to the tubes Invert the tubes back and forth twice Vortex the tubes for 5 seconds at room temperature at setting 7 IMPORTANT If the magnetic particles stick to the tube wall flush gently with the Wash Solution to detach the particles b Quick spin the tubes in a microcentrifuge for 15 seconds then place th
8. USER GUIDE applied biosystems by Life technologies PrepSEQ Residual DNA Sample Preparation Kit for use with resDNASEQ Quantitative E coli DNA kit resDNASEQ Quantitative Vero DNA kit Publication Part Number 4460663 Rev B Revision Date May 2011 6 technologies For research use only Not intended for any human or animal therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES Limited Use Label License Environmental Food Agricultural Testing amp Quality Control The purchase price of this product includes a limited non transferable immunity from suit under U S Patents and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche and under U S Patents and corresponding patent cla
9. ZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals guidelines Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About SDSs on page 22 Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 21 Safety Appendix Chemical waste safety Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal About SDSs Chem
10. col Select the program labeled PrepSEQ resDNA NE 30 from the MagMax Express 96 Load the plates Step 1 Press START then position the plates according to Comb loading plate the Display window instructions a Elution plate with 100 uL or 200 pL elution buffer b Wash 2 plate with 300 uL wash buffer c Wash 1 plate with 300 uL wash buffer d Lysis plate D 0 me rm O LD e 3 2 D ay 0 e o EX m o 2 Step 2 Place the plates in the instrument and load them in the following order pressing START to move the turntable for each plate y Prepare samples for digestion Step 1 Add 100 uL of sample to a well of the 96 deep well a Step 3 Make a master mix of Proteinase K buffer and plate Adjust pH level to between 6 and 8 first using 10 N Proteinase K then add 70 uL to each sample Vortex and NaOH or 10 N HCL if necessary then measure and confirm spin briefly Incubate the mix at 56 C for 30 minutes the pH level v Y Step 4 Place the plate in the processor then press START to begin the lysis process Step 2 Optional Add 10 uL of 5 M NaCl if the salt concentration of the sample is lower than 0 5 M see page 17 The instrument mixes the samples for 10 seconds at fast J speed then incubates the samples at 57 C for 30 minutes mixing at slow speed Prepare the lysis and bind the DNA Step 1 Incubate the Magnetic Particle suspension at Step 4 Add 300 UL of Binding Solution
11. contents and The PrepSEQ Residual DNA Sample Preparation Kit contains the PrepSEQ Nucleic storage Acid Extraction Kit Part no 4400799 which contains three component boxes and the PrepSEQ Residual DNA Sample Preparation Kit Part no 4399042 Kit components include Reagent Description Storage Part Number PrepSEQ Nucleic Acid Extraction Kit Box 1 4400793 Lysis Buffer 2 bottles 50 mL bottle Store at room temperature 4400659 Binding 1 empty bottle NA 4400789 Solution Isopropanol Wash Buffer 2 bottles 26 mL bottle Store at room temperature 4400783 Concentrate Elution Buffer 1 bottle 25 mL Store at room temperature 4400784 Proteinase K 1 bottle 50 mL Store at room temperature 4400787 PK Buffer PrepSEQ Nucleic Acid Extraction Kit Box 2 4400795 Magnetic 2 tubes 1 5 mL tube Store at 2 8 C 4401405 Particles PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 7 PrepSEQ Residual DNA Sample Preparation Kit Protocol Required materials and preparation Reagent Description Storage Part Number PrepSEQ Nucleic Acid Extraction Kit Box 3 4400675 Proteinase K 1 tube 20 mg mL 1 25 mL Store at or below 20 C 4403958 PrepSEQ Residual DNA Sample Preparation Kit 4399042 Proteinase K 1 tube 20 mg mL 1 25 mL Store at or below 20 C 4403958 Yeast tRNA 1 tube 10 mg mL 0 5 mL Store at or below 20 C 4404626 Glycogen 2 tubes 5 mg mL 1 0 mL tube Store at or below
12. e tubes in the magnetic stand and let the tubes stand for 1 minute Note The magnetic particles with the bound DNA are magnetically captured after approximately 1 minute c Without disturbing the magnetic beads remove the supernatant using a Pipetman or by aspiration d Remove tubes from the magnetic stand then add 300 uL of Wash Solution to the tube Invert the tubes back and forth twice Vortex the tubes for 5 seconds at room temperature at setting 7 a 0 o n m O nN ied 3 Ig 0 P 0 o L E e j IMPORTANT If the magnetic particles stick to the tube wall flush gently with the Wash Solution to detach the particles e Quick spin the tube in a microcentrifuge for 15 seconds then place the tubes in the magnetic stand and let the tubes stand for 1 minute Note The magnetic particles with the bound DNA are magnetically captured after approximately 1 minute f Without disturbing the magnetic beads remove the supernatant using a Pipetman or by aspiration g Use a P200 to remove the residual solution from the bottom of the tube h With the tube lid open air dry the magnetic particles pellet in the magnetic stand for 5 minutes at room temperature IMPORTANT Air drying removes ethanol from the Wash Solution Ethanol decreases recovery and inhibits PCR 4 Elute the DNA a Add 50 uL of Elution Buffer to each tube containing magnetic particles that have bound DNA IMPORTANT Th
13. e magnetic particles may be difficult to detach from the tube wall Place the tube in the microcentrifuge with the magnetic particles pellet oriented toward the center then spin the tube for 30 seconds to detach the magnetic particles from the tube wall into the Elution Buffer If the magnetic particles are difficult to resuspend use a P200 to gently pipet up and down several times Be careful not to let the magnetic particles stick inside the pipette tip b Vortex the tubes for 10 seconds at high speed then incubate the tubes at 70 C for 7 minutes Vortex the tubes two to three times during the incubation to help resuspension PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 15 PrepSEQ Residual DNA Sample Preparation Kit Protocol Automated protocol for residual DNA extraction C Centrifuge the tubes in a microcentrifuge for 15 seconds place the tubes in the magnetic stand and let the tubes stand for 2 minutes Then transfer the liquid phase containing the eluted DNA to a new nonstick 1 5 mL microcentrifuge tube Note The magnetic particles are magnetically captured in approximately 1 minute DNA is in the eluate Centrifuge the tube at top speed 215 000 x g for 3 minutes to pellet the residual magnetic particles then place the tube in the magnetic stand and let the tube stand for 1 minute Transfer the liquid phase containing the eluted DNA to the nonstick 1 5 mL microcentrifuge tube without disturbing the ma
14. gnetic particles Use 10 uL of the eluate in the real time PCR Note Magnetic particles can inhibit PCR When you finish the sample extraction procedure refer to the resDNASEQ Quantitative DNA Kits Protocol Part no 4415260 to set up PCR for DNA quantitation Automated protocol for residual DNA extraction Prepare for automated sample preparation of E coli and Vero DNA 16 You can use the MagMAX Express automation platform to automate the extraction of host cell line residual DNA Perform the steps in the following protocol for automated extraction For all chemicals read the Safety Data Sheet SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves 1 Prepare the plates Plate name Plate type Sample or buffer added Lysis 96 deep well plate 100 uL sample 60 uL PK buffer 10 uL PK Wash 1 96 deep well plate 300 uL Wash buffer Wash 2 96 deep well plate 300 uL Wash buffer Elution 96 deep well plate 100 or 200 uL Elution buffer Comb loading 96 deep well tip comb combined plate with 96 standard plate 2 Select the instrument protocol Select the program labeled PrepSEQ_ResDNAv1 from the MagMax Express 96 3 Load the plates Load plates into the instrument in the order listed here After loading each plate press START to move the turntable a b C Comb loading plate Elution plate with 100 uL or 200 uL buffer Wash 2 plate with 3
15. he DNA a Incubate the Magnetic Particle suspension at 37 C for 10 minutes then vortex for 2 minutes or until completely suspended b Remove the plate from the instrument then add 360 uL of Lysis Solution using an 8 channel pipette The Lysis Solution will be freshly supplemented with Glycogen and with Yeast tRNA at final concentrations of 8 ug uL and 10 ug uL respectively as described on page 11 Pipet up and down two times to mix c Add 30 ul of Magnetic Particle suspension to the sample then shake the plate gently to mix d Add 300 ul of Binding Solution using an 8 channel pipette then pipet up and down three times to mix e Place the plate back into the instrument loading position then press START to begin binding The instrument mixes the beads for 15 minutes superfast speed collects beads 45 counts then washes and elutes the DNA The comb with beads will automatically be washed in the Wash 2 plate and placed on the comb loading plate Then the Elution plate will be automatically placed into its loading position and the eluates will be ready for analysis 6 Measure the eluate volume a Place the Elution plate on a Magnetic Stand 96 to attract residual particles to the bottom of the wells b Use a pipette to measure the eluate volume from several wells eluate volumes can be homogeneous The average eluate volume is used to calculate recovery efficiency PrepSEQ Residual DNA Sample Preparation Kit Use
16. ical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood e After emptying a waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure the health and safety of all personnel in your laboratory Ensure that the instrument waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 23 Safety Appendix Biological hazard safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipme
17. ical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files Obtaining SDSs The SDS for any chemical supplied by Life Technologies Corporation is available to you free 24 hours a day To obtain SDSs 1 Goto www appliedbiosystems com click Support then select SDS 2 In the Keyword Search field enter the chemical name product name SDS part number or other information that appears in the SDS of interest Select the language of your choice then click Search 3 Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you select Note For the SDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Chemical waste safety Chemical waste T WARNING HAZARDOUS WASTE Refer to Safety Data Sheets SDSs and hazards local regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Appl
18. ied Biosystems instruments are potentially hazardous and can cause injury illness or death 22 PrepSEQ Residual DNA Sample Preparation Kit User Guide Safety Appendix Chemical waste safety WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chem
19. ims outside the United States owned by Applied Biosystems for using only this amount of product in the practice of the 5 nuclease process solely in environmental testing quality control quality assurance testing food and agricultural testing including reporting results of purchaser s activities in environmental testing quality control quality assurance testing food and agricultural testing for a fee or other commercial consideration and also for the purchaser s own internal research and development activities No right under any other patent claims such as apparatus or system claims and no right to perform the 5 nuclease process for any other purpose is hereby granted expressly by implication or by estoppel This product is for environmental testing quality control quality assurance testing food and agricultural testing and research purposes only Human diagnostic uses require a separate license from Roche Further information regarding 5 nuclease licensing program and commercial service licenses may be obtained by contacting outlicensing alifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners TagMan and AmpliTaq Gold are registered trademarks of Roche Molecular Systems Inc PIPETMAN is a registered trademark of Gilson Medical Electronics 2011 Life Technologies Corp
20. in death or serious injury This signal word is to be limited to the most extreme situations The SDSs for any chemicals supplied by Life Technologies Corporation or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see the Safety Appendix on page 21 IMPORTANT For the SDSs of chemicals not distributed by Life Technologies Corporation or Ambion contact the chemical manufacturer PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 5 About This Guide How to use this guide How to use this guide Text conventions This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open Spot Set Right click the sample row then select View Filter K View All Runs User attention Two user attention words appear in Life Technologies Corporation user words documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument ope
21. n new assays and updated 7500 product documentation go to www microseq com For information on performing PCR after sample extraction refer to the resDNASEQ Quantitative DNA Kits Protocol Part no 4460662 For information on MagMAX Express 96 DW instrument see the Applied Biosystems MagMAX Express 96 User Manual Part no N07849 Portable document format PDF versions of this guide and the document shown above are available at www appliedbiosystems com Note To open the documentation available from the Life Technologies Corporation web site use the Adobe Acrobat Reader software available at www adobe com How to obtain support For the latest services and support information for all locations go to www appliedbiosystems com At the Life Technologies Corporation web site you can Access worldwide telephone and fax numbers to contact Life Technologies Corporation Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Life Technologies Corporation user documents SDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 25 ill Headquarters 5791 Van Allen Way Carlsbad CA 2008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 F
22. nt which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at http www cdc gov biosafety publications index htm e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov Chemical alerts General alerts for all chemicals 24 Avoid contact with skin eyes and or clothing Read the SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves PrepSEQ Residual DNA Sample Preparation Kit User Guide Documentation Related documents For additional documentation see How to obtain support below For brief instructions on using the PrepSEQ Residual DNA Sample Preparation Kit see the PrepSEQ Residual DNA Sample Preparation Kit Quick Reference Part no 4468125 For information o
23. ntation sss kk kk kk kk kk kk KK REOR kk kk kk kk kk kk lk 25 Related documents n n kk kk kk kk kk kk kk kk kk kk kk kk kk kk n 25 How to ObtainiiSup Ports cca hice ven eR Ut RR EIER seite sided 25 PrepSEQ Residual DNA Sample Preparation Kit User Guide 3 Contents 4 PrepSEQ Residual DNA Sample Preparation Kit User Guide About This Guide Safety information Safety alert words SDSs Note For general safety information see this Preface and Safety Appendix When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Four safety alert words appear in Life Technologies Corporation user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical T CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result
24. on to approximately 0 5 M if necessary Y Step 6 Make a master mix of Proteinase K buffer and Proteinase K then add 70 uL of Proteinase K buffer Proteinase K to the sample per 100 uL of sample Briefly vortex and spin Incubate at 56 C for 30 min Y Step 7 Add 360 uL of lysis solution mix per 100 uL of starting sample Y Bind DNA Step 1 Add 30 uL of Magnetic Particles using a wide bore pipette tip Y Step 2 Add 300 uL of Binding Solution per 100 uL of starting sample invert twice then vortex for 5 min at setting 7 Step 3 Spin for 15 seconds place the tubes into a magnetic stand for 5 min or until the solution is clear then remove and discard the supernatant Y Wash DNA Step 1 Remove tubes from the magnetic stand then add 300 uL of Wash Solution Invert the tubes twice Vortex for 5 sec at setting 7 see the corresponding IMPORTANT on page 15 Step 2 Spin for 15 sec then place the tubes into the magnetic stand for 1 min see the corresponding Note on page 15 Y Step 3 Without disturbing the magnetic beads remove and discard the supernatant using a Pipetman or by aspiration Step 4 Remove tubes from the magnetic stand then add 300 UL of Wash Solution Invert the tubes twice Vortex for 5 sec at setting 7 see the corresponding Note on page 15 pe Step 5 Spin for 15 sec then place the tubes into the magnetic stand for 1 min see the corresponding Note on page 15 Y
25. or support visit www appliedbiosystems com support technologies www lifetechnologies com
26. oration All rights reserved 4460663 Rev B Contents About This Guide lll x x kk kk kk kk kk rent mx kk kk kk kk 5 Safety inforimalion cse cess eh AWA e LENS edema qe e UR UR e er lens 5 How to use this guide MM kk kk kk kk kk kk kk kK kK KK kk kK rn 6 PROTOCOL PrepSEQ Residual DNA Sample Preparation Kit Protocol 7 Product overview a kak eb ERR RE ed EA ER Ric n dR ROK 7 Required materials and preparation MAW kk kk kk kk kk kk cece cece KK KK KK KK KK KK KK KK KK 7 Extract Host GellDNA 3 54 GZ Sale BSS EU alay M eI UMEN prb 11 Workflow for manual residual DNA extraction WWW kk kk kk kk kk KK KK KK KK KE RR KK KK KK 12 Workflow for automated E coli and Vero DNA extraction n nana kk kk KK KK KK KK 13 Manual protocol for residual DNA extraction kk kk KK KK KK KK KK KK KK KK KK KK 14 Automated protocol for residual DNA extraction kk kk kk kK KK KK KK KK KK KK KK KK 16 Dilute DNA samples if necessary Jk kk kk kk kk kk kk KK KK KK KK KK KK KK e 18 Troubleshooting t eI ERU Du ule Dus E el soit ROM onu A atero 19 APPENDIX A Safety Appendix iis ee x x cc kk i dei kk kk kk kk kk kk Ou EE 21 Chemical Safety 2 2 20 aka Sakan h na bada h ee del e IR EID Hope ce EUER nas 21 Chemical waste safety 0 0 0 cece cece n 22 Biological hazard safety kk kk kk kk KK KK KK KK KK KK KK kK kk kk e eee 24 Chemical al Sy arria saa Ay wap ne s ne del cede YE ERIT Ak ad e haber eee rds 24 Docume
27. ortex the Magnetic Particles at setting 7 until resuspension is complete Label 2 mL Safe Lock tubes as appropriate then add 100 or 200 uL samples into each tube Note Test samples from the early purification process often contain levels of DNA that are above the highest point of the residual DNA assay standard curve See Dilute DNA samples if necessary on page 18 Alternatively make dilutions of the extracted DNA before running the PCR reaction Add an Extraction Negative Control use 100 uL or 200 uL DNA Dilution Buffer as sample Measure the pH of the solution with pH paper If necessary add sufficient 10 N NaOH or 10 N HCI to adjust the pH of the solution to between 6 and 8 Note Alternatively adjust the pH by adding the 10 N NaOH or 10 N HCI before preparation using an appropriately smaller amount For example if 20 uL of 10 N NaOH adjusts the pH of 1 mL of sample then add 2 uL per 0 1 mL of 10 N NaOH before preparing the sample Adjust salt concentration in the samples with 5 M NaCl to approximately 0 5 M if the salt concentration is lower than 0 5 M For example add 10 uL of 5 M NaCl per 100 uL of sample Make a master mix of Proteinase K buffer and Proteinase K then add 70 uL of Proteinase K buffer Proteinase K per 100 uL of sample Briefly vortex and spin Incubate at 56 C for 30 minutes If only one block heater is available after incubation reset it to 70 C for the elution step Add 360 u
28. r Guide 17 D 0 gel GH m O LD 5 3 jel D D 0 o e lt E e 2 PrepSEQ Residual DNA Sample Preparation Kit Protocol Dilute DNA samples if necessary C Use a multi channel pipette to carefully transfer 10 uL of eluate into the PCR reaction plate for the real time PCR assay Do not touch the particles Dilute DNA samples if necessary 18 Test samples from the early purification process often contain levels of DNA that are above the highest point of the residual DNA assay standard curve You must dilute these samples from 1 100 up to 1 10 000 prior to PrepSEQ Residual DNA sample preparation Diluting samples in water is often not efficient because the PrepSEQ Residual DNA Sample Preparation Kit protocol is optimized for highly efficient recovery of DNA from complex mixtures of proteins buffer and salts To rectify this situation dilute test samples prior to DNA extraction and purification with a solution of 1X PBS pH 7 4 free of Mg and Ca plus 0 5 M NaCl 1X PBS can be made by diluting Ambion 10 XPBS Part no AM9624 If the sample is being diluted use the sample dilution buffer as the negative process control instead of water Alternatively dilute extracted DNA before running the PCR reaction PrepSEQ Residual DNA Sample Preparation Kit User Guide Troubleshooting PrepSEQ Residual DNA Sample Preparation Kit Protocol Troubleshooting Observation Possible cause Action
29. ration accurate chemistry kit use or safe use of a chemical 6 PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Product overview The PrepSEQ Residual DNA Sample Preparation Kit extracts host cell DNA from products produced in cell lines such as E coli and Vero The kit uses chemical lysis and magnetic beads to efficiently extract genomic DNA from diverse sample types including samples that contain high protein and low DNA concentration To assure accurate quantitative results Applied Biosystems protocols call for true triplicate sample preparation and analysis Extract each test sample in triplicate and perform a single PCR reaction for each extraction The instrument software calculates a mean quantity You can calculate the percent coefficient of variation from this data SD Mean Quantity x 100 CV Based on this calculation you can then assign a CV to ensure accurate results from each sample tested z0 0 o nD rm O LD o 3 je 0 RU 0 O o m o 2 After extraction you can quantitate the DNA to determine the level of host cell DNA contamination in the product For quantitation of host cell line residual DNA Applied Biosystems recommends use of the resDNASEQ Quantitative DNA kits as described in the resDNASEQ Quantitative DNA Kits Protocol Part no 4415260 Required materials and preparation Kit
30. the bottle to indicate that it contains ethanol then store the bottle at ambient temperature PrepSEQ Residual DNA Sample Preparation Kit User Guide 9 PrepSEQ Residual DNA Sample Preparation Kit Protocol Required materials and preparation Proteinase K Proteinase K Buffer mix Prepare a mix that contains Proteinase K and Proteinase K buffer for the total number of samples to be processed Include a 10 overage to account for pipetting losses For example if you have 9 samples create a mix for 10 samples as shown in the following table Then add 70 uL of the mix per 100 uL of sample 1 reaction 10 reactions Component per 100 pL of per 100 pL of sample sample Proteinase K 10 uL 100 uL Proteinase K buffer 60 uL 600 uL Magnetic particles 1 Immediately before using incubate the tube containing the magnetic particles at 37 C for 10 minutes 2 If necessary use a P1000 Pipetman to agitate the particles at the bottom of the tube before vortexing Small aggregations of particles will reduce performance 3 Vortex the tube at setting 7 to completely resuspend the particles 10 PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit Protocol Extract Host Cell DNA Lysis Solution Mix of Lysis Buffer tRNA and glycogen prepared immediately prior to starting sample preparation Prepare a fresh mixture according to the following table Use 360 uL of
31. the mix for sample preparation per 100 uL of sample Reagent Volume uL Glycogen 5 mg mL 180 uL 3 tRNA 10 mg mL 4 uL 8 Lysis buffer 7600 uL e Total 7784 uL a o le 3 D S j Extract Host Cell DNA To extract host cell DNA from products that are produced in cell lines such as E coli or Vero use the following workflow For all chemicals read the Safety Data Sheet SDS and follow the handling instructions Wear appropriate protective eyewear clothing and gloves PrepSEQ9 Residual DNA Sample Preparation Kit User Guide 11 PrepSEQ Residual DNA Sample Preparation Kit Protocol Extract Host Cell DNA Workflow for manual residual DNA extraction The manual extraction workflow is shown below For details go to page 14 Prepare digestion reaction tubes and Proteinase K reaction Step 1 Incubate the Magnetic Particles at 37 C for 10 min then vortex the Magnetic Particles at 7 to completely resuspend particles Y Step 2 Label 2 mL Safe Lock tubes as appropriate then add 100 or 200 uL of sample to each tube Sample dilution may be needed see page 18 Y Step 3 Add an Extraction Negative Control use 100 uL or 200 uL DNA Dilution Buffer as sample Y Step 4 Adjust pH level to between 6 and 8 first using 10 N NaOH or 10 N HCI if necessary then measure and confirm i the pH level The required volume depends on the sample pH Step 5 Adjust NaCl concentrati
32. using an 37 C for 10 min then vortex for 2 min or until completely 8 channel pipette then pipet up amp down two times suspended Y Step 5 Place the plate back into the instrument loading y s Step 2 Remove the plate from the instrument then add position then press START to begin binding Y 360 uL of Lysis Solution using a multi channel pipette Pipet up amp down two times to mix P The instrument mixes the beads for 15 min superfast d collects beads 45 ts th h d Step 3 Add 30 ul of Magnetic Particle suspension to the m DNA ead Se counts den Weenies al sample then shake the plate gently to mix Measure the eluate volume Step 1 Place the Elution plate on a Magnetic Stand 96 to attract residual particles to the bottom of the wells Step 2 Use a pipette to measure the eluate volume from several wells eluate volumes can be heterogeneous The average eluate volume is used to calculate recovery efficiency Step 3 Use a multi channel pipette to carefully transfer 10 uL of eluate to the PCR reaction plate Do not touch particles PrepSEQ Residual DNA Sample Preparation Kit User Guide 13 PrepSEQ Residual DNA Sample Preparation Kit Protocol Manual protocol for residual DNA extraction Manual protocol for residual DNA extraction 1 Prepare the digestion reaction tubes and Proteinase K reaction a b Set a block heater to 56 C Incubate the Magnetic Particles at 37 C for 10 minutes then v
Download Pdf Manuals
Related Search