PrepSEQ® Residual DNA Sample Preparation Kit User Guide (Pub
Contents
1. Automated protocol for residual DNA extraction 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 MagMAX Express requires the following 5 plates Plate name Plate type Lysis 96 deep well plate Wash 1 96 deep well plate Wash 2 96 deep well plate Elution 96 deep well plate Comb loading plate 96 deep well tip comb combined with 96 standard plate Perform these steps for automated DNA extraction 1 Prepare the Wash 1 Wash 2 and Elution plates according to the following table Plate name Plate type Volume of buffer to add Wash 1 96 deep well plate 300 UL of Wash buffer Wash 2 96 deep well plate 300 UL of Wash buffer Elution 96 deep well plate 200 uL of Elution buffer 2 Prepare samples for PK digestion Lysis plate 16 PrepSEQ Residual DNA Sample Preparation Kit User Guide Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Automated protocol for residual DNA extraction 3 Ifnecessary adjust the sample pH to between 6 and 8 using 10 N NaOH or 10 N HCI Use pH paper to confirm the sample pH Note Alternatively adjust the pH by adding the 10 N NaOH or 10 N HCI befor2. 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 equipment 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 In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html 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 In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Hea
3. idelines for bestylelds sa tn 3 eh ai hee teh SAL Ne uh ah ale a eit oceans 14 Manual protocol for residual DNA extraction 0c c cece KK KK KK KK KK KK KK eens 14 Prepare and digest the samples 0 cece cece KK KK KK ee eee KK kk kk kk kk kk 14 BIG es Aesth O E Pee E cpt edt ach titan BEY AN Gols De estes 15 Washthe DNA stats cA td aceite n Hea ties 15 Blute the SS 2ee Ie eDeDererreE IePePeERPEREEFErearererearaa rrr r r braco lec a 15 Automated protocol for residual DNA extraction 00 c eee e cece eee eee KK KK KK 16 Troubleshooting nai iaten std do hh A eee a a hd K o a a ates el eet th ae 18 APPENDIX A Good Laboratory Practices lt e KK 19 Residual DNA quantification kk kk kk kk kk kk kk kk kK KK KK KK KK kK KK kk kk kk kk kk kk kk kk kk kk ka 19 Diluting test samples for residual DNA sample preparation KK 19 Preparing serial dilutions and a standard curve WWW rr 19 PrepSEQ Residual DNA Sample Preparation Kit User Guide 3 Contents Preventing PCR contamination kk kk kk kk kk kK KK KK KK KK KK KK kK kk kK kk kk kk kk kk kk ka 19 Avoiding false positives due to cross contamination WWW kk kk kk KK KK KK KK KK KK 20 Plate layout suggestions vii k la keka a A nie kal din sen ges W RAA a n erk 20 APPENDIX B Safety aaa kk kk kk kK KK KK kk kk KK KK kk kk kk kk 21 B RA ST VB DD o o H HHHH HHHH ee 22 Specific chemical handling 02000 kk kk kK kK KK KK KK KK KK KK kK KK kk kk
4. non transferable right to use the purchased amount of the product la to perform internal research for the sole benefit of the purchaser and b for 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 No other right 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 The purchase of this product does not grant the purchaser any additional rights including without limitation the right to transfer or resell the product in any form or the right to use the product as a therapeutic agent or diagnostics test component For information on obtaining additional rights please contact outlicensingfalifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc TaqMan used under permission and license Microsoft and Windows are registered trademarks of Microsoft Corporation 2014 Thermo Fisher Scientific Inc All rights reserved Con
5. Residual DNA Sample Preparation Kit User Guide About This Guide Prerequisites 6 PrepSEQ Residual DNA Sample Preparation Kit User Guide PrepSEQ Residual DNA Sample Preparation Kit M Product description sian e a ai is 7 E Kit contents and storage rr 8 B Regquiredimatertals a aaa ai 9 S Workflow for residual DNA extraction manual or automated from CHO E coli Human Vero Pichia NSO and MDCK oo 11 m Prepare reagents Soyani oh set li tds dates 11 E Guidelines for best yields a a R R TER EN eee a3 k wa Aw 14 S Manual protocol for residual DNA extraction 0 00 c eee eee eee 14 m Automated protocol for residual DNA extraction K3 16 m Dilute test samples if necessary eee KK KK KK KK 12 E Troubleshooting a aio 18 Product description The PrepSEQ Residual DNA Sample Preparation Kit extracts host cell DNA from products produced in cell lines such as CHO E coli Human Vero Pichia NSO and MDCK 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 ensure accurate quantitative results Life Technologies 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 coeff
6. kk kk kk kk ka 23 Biological hazard safety WW kk kk kk kk kk kK KK kk KK KK KK KK KK kK kk kk kk kk kk kk kk kk kk kk kk ka 23 Documentation and Support xx kk kk kk KK KK KK KK KK kk kk kk kk kk 25 Related documentation oinean EEN ESRR mm mm k z 25 Obtaining SDSS aa 0 PO oe coher soleil oil ei ele iy tae de cd 25 Obtaining SUPPORT tt Gal eee te 25 Limited product Warranty besa pve a kk kk kk kk kk ka 26 PrepSEQ Residual DNA Sample Preparation Kit User Guide About This Guide IMPORTANT Before using this product read and understand the information in the Safety appendix in this document Revision history Revision Date Description B January 2013 Add the following kits e resDNASEQ Quantitative CHO DNA Kit Cat no 4402085 e resDNASEQ Quantitative E coli DNA Kit Cat no e 4458435 e resDNASEQ Quantitative Vero DNA Kit Cat no 4458444 December 2014 Add the resDNASEQ Quantitative Human DNA Kit Cat no A26366 Purpose This guide provides step by step instructions for using the PrepSEQ Residual DNA Sample Preparation Kit to efficiently extract genomic DNA from diverse sample types including samples that contain high protein and low DNA concentration Prerequisites This guide uses conventions and terminology that assume a working knowledge of the Microsoft Windows operating system the Internet and Internet based browsers PrepSEQ
7. or brown precipitate may form in the Magnetic Particles tube if it is stored at 2 8 C The precipitate will dissolve when it is heated to 37 C for a minimum of 10 minutes with intermittent vortexing Make sure the precipitate is completely dissolved before using the beads Manual protocol for residual DNA extraction Prepare and digest the samples 14 1 2 Set a block heater to 56 C If available set a second block heater to 70 C If necessary adjust the sample pH to between 6 and 8 using 10 N NaOH or 10 N HCI Use pH paper to confirm the sample pH 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 processing the sample Label 2 mL Safe Lock tubes as appropriate then add 100 uL or 200 uL of sample 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 test samples if necessary on page 12 Alternatively make dilutions of the extracted DNA before running the PCR reaction Add 10 uL of 5 M NaCl per 100 uL of sample Add 70 uL of Proteinase K buffer Proteinase K mix see Prepare reagents on page 11 per 100 uL of sample Briefly vortex and spin Incubate at 56 C for 30 minutes If only o
8. the tube wall during the elution step 1 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 Do not overdry Magnetic Particles are difficult to resuspend during the elution step 2 on page 16 Incubate the pellets at 70 C for 7 minutes Vigorously vortex the tubes three times during incubation to help resuspension Do not overdry Formation of precipitate in Magnetic Particles page 16 Incubate the Magnetic Particle suspension at 37 C for a minimum of 10 minutes with intermittent vortexing at setting 7 or until the particles are completely suspended Particles no longer produce consistent Samples have low pH step 2 on page 14 Measure the pH of the sample and adjust the pH to between 6 and 8 results fine brown sandy particles and brown color in the supernatant Magnetic Particles were stored at 20 C Kit contents and storage Magnetic Particles on page 8 Order new materials and store them at room temperature 18 PrepSEQ Residual DNA Sample Preparation Kit User Guide Good Laboratory Practices Residual DNA quantification lt eee es 19 Preventing PCR contamination KK kk KK KK KK KK KK KK KK KK KK KK KK 19 Plate layout suggestions WW kK kk KK KK KK KK KK KK KK KK KK K
9. 0027 PrepSEQ Residual DNA Sample Preparation Kit User Guide Required materials not included in the kit Required materials 10 Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Item Source or Cat no Equipment Block heater for use with 2 mL tubes Manual DNA extraction involves two incubations at different settings so two heaters may be convenient Major laboratory supplier MLS Magnetic stand 16 position 4457858 Benchtop microcentrifuge for 1 5 mL and 2 mL tubes MLS 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 Pipetman Pipettors P1000 P200 P20 and P10 MLS Positive displacement e Air displacement e Multichannel Nonstick RNase free Microfuge Tubes 1 5 mL AM12450 1 box 250 tubes box Safe Lock PCR clean microcentrifuge tubes round bottom 2 mL VWR 62111 754 Reagents Ethanol 95 MLS IMPORTANT Do not use denatured ethanol It contains components that are not compatible with the protocol 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 needed to AM9624 dilute samples PrepSEQ Residual DNA Sample Preparation Kit User Guide Chapter 1 PrepSEQ Residual DNA Sample Prepar
10. 014
11. 21 Appendix B Safety Chemical safety Chemical safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions 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 To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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 After emptying a waste container seal it with the cap provided Characterize by analysis i
12. A sample preparation Diluting samples in water or TE will reduce extraction efficiency The PrepSEQ Residual DNA Sample Preparation Kit protocol is optimized to obtain highly efficient recovery of DNA from complex mixtures of proteins buffer and salts For best results dilute test samples before DNA extraction with a solution of 1X PBS pH 7 4 free of Mg and Ca 1X PBS can be made by diluting 10X PBS Cat no AM9624 If you are diluting the sample use the sample dilution buffer as the Negative Process Control instead of water Alternatively dilute extracted DNA before running the PCR reaction Prepare triplicate reactions 12 To ensure accurate quantitative results Life Technologies protocols call for true triplicate sample preparation and analysis You must extract each test sample in triplicate and perform a single PCR reaction for each extraction Note After PCR the instrument software calculates a mean quantity and a standard deviation for the triplicate samples You can calculate the percent coefficient of variation from this data SD Mean Quantity x 100 CV PrepSEQ Residual DNA Sample Preparation Kit User Guide Recommended Prepare the extraction controls Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Prepare triplicate reactions In addition we recommend that you prepare the following extraction controls for each test sample Three negative extraction controls NEG The NEG i
13. K KK KK KK k 20 Residual DNA quantification Diluting test samples for residual DNA sample preparation Preparing serial dilutions and a standard curve Test samples from early in the purification process often contain levels of DNA that are above the highest point on the standard curve of the residual DNA assay Dilute the samples appropriately from 1 100 up to 1 10 000 prior to sample preparation The PrepSEQ sample preparation protocol is optimized for highly efficient recovery of DNA from complex mixtures of proteins buffer and salts Because of this recovery of DNA from samples diluted in water or TE is often not efficient We recommend e 50mMTris pH 8 0 0 5 M NaCl or e PBS Follow these guidelines when you prepare serial dilutions with the standard DNA provided in the kit to generate the standard curve for quantitation of DNA in test samples e Prepare dilutions in nonstick 1 5 mL microfuge tubes Cat no AM12450 e Prepare the standard curve and the test samples in different areas of the lab Use different sets of pipettors for test sample preparation and for standard curve preparation and aliquoting to avoid cross contamination of test samples e Vortex each tube to mix the contents thoroughly before each dilution step e Use DNA Dilution Buffer DDB for all dilutions of Standard DNA Label the top of each tube as indicated in the protocol You can store the standard curve preparation at 2 8 C fo
14. RC sample containing 10 pg of DNA control per well for qPCR analysis e ERC10pg Add 16 7 uL from tube SD3 to 100 uL test samples in triplicate Tube SD3 is the 3 pg uL standard dilution tube from the standard curve prepared earlier refer to the resDNASEQ Quantitative DNA Kits User Guide Pub no 4469836 3 Vortex each tube thoroughly to mix 4 Proceed to DNA extraction using the manual protocol page 14 or automated protocol page 16 PrepSEQ Residual DNA Sample Preparation Kit User Guide 13 Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Guidelines for best yields Guidelines for best yields e Maintain a homogenous suspension of the magnetic beads because this maximizes the surface area to which the DNA can bind The appearance of the mixture should be homogenous after mixing Once dried the DNA will stay bound to the magnetic beads Do not allow the magnetic beads to over dry because this reduces the elution efficiency over dried beads are not easily resuspended During manual elution vortex every 2 minutes to assist elution This will result in better yield during recovery Note During washing steps it is not necessary to detach the Magnetic Particles from the tube wall Although some test samples cause the beads to adhere very firmly to the tube wall while others form loose pellets that detach during the vortex steps all pellets will dissolve with vortexing during heated elution Note White
15. USER GUIDE applied biosystems by HE technologies PrepSEQ Residual DNA Sample Preparation Kit For use with resDNASEQ Quantitative CHO DNA kit resDNASEQ Quantitative E coli DNA kit resDNASEQ Quantitative Human DNA kit resDNASEQ Quantitative Vero DNA kit resDNASEQ Quantitative Pichia DNA kit resDNASEQ Quantitative NSO DNA kit resDNASEQ Quantitative MDCK DNA kit Publication Number 4469838 Revision C o For Research Use Only Not for use in diagnostic procedures technologies For Research Use Only Not for use in diagnostic procedures The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 Limited Use Label License No 492 Environmental Testing Quality Control Quality Assurance Testing Food and Agricultural Testing Notice to Purchaser The purchase of this product conveys to the purchaser the limited
16. amplification Use different sets of pipettors when pipetting negative control unknown and positive control samples Plate layout suggestions 20 For each plate row dispense in sequence from left to right the negative controls unknown samples and positive controls at the end of the row or column Place positive controls in one of the outer columns If possible separate all samples from each other by at least one well if space is limiting place at least one well between unknown samples and controls Be aware that caps come in strips of 8 or 12 PrepSEQ Residual DNA Sample Preparation Kit User Guide Safety WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document PrepSEQ Residual DNA Sample Preparation Kit User Guide
17. ants and wash buffers during sample prep we recommend attaching the waste collection bottle to the vacuum using tubing that can accommodate 200 uL pipette tips 1 Elute the DNA 1 Remove the tubes from the magnetic stand then add 300 uL of Wash Solution to the tubes Vortex the tubes for 5 seconds at room temperature at setting 7 Quick spin the tubes in a microcentrifuge at top speed 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 Without disturbing the magnetic beads remove the supernatant using a Pipetmar or by aspiration Remove the tubes from the magnetic stand then add 300 uL of Wash Solution to each tube for a second wash Vortex the tubes for 5 seconds at room temperature at setting 7 Quick spin the tubes in a microcentrifuge at top speed for 15 seconds then place the tubes in the magnetic stand for 1 minute Note The Magnetic Particles with the bound DNA are magnetically captured after approximately 1 minute Start the 5 minute timer Without disturbing the magnetic beads remove the supernatant using a Pipetman or by aspiration Use a P200 to remove the residual solution from the bottom of the tube With the tube lid open air dry the Magnetic Particles pellet in the magnetic stand for no more than 5 minutes at room temperature IMPORTANT Air dry to r
18. ation Kit 1 Workflow for residual DNA extraction manual or automated from CHO E coli Human Vero Pichia NSO and MDCK Workflow for residual DNA extraction manual or automated from CHO E coli Human Vero Pichia NSO and MDCK Workflow for automated residual DNA extraction on Workflow for manual residual DNA extraction the MagMAX Express 96 DW instrument Prepare reagents page 11 Prepare reagents page 11 Dilute test samples if necessary page 12 Dilute test samples if necessary page 12 Prepare triplicate reactions page 11 Prepare triplicate reactions page 11 Prepare and digest the samples page 14 Prepare the Wash 1 Wash 2 and Elution plates page 16 Bind the DNA page 15 Prepare samples for PK digestion Lysis plate page 16 Wash the DNA page 15 Load the plates into the instrument page 17 Elute the DNA page 15 Add additional components to the Lysis plate page 17 Prepare reagents Before you use the PrepSEQ Residual DNA Sample Preparation Kit prepare the following solutions Prepare magnetic 1 Set a block heater to 37 C beads 2 Incubate the Magnetic Particle suspension at 37 C for a minimum of 10 minutes with intermittent vortexing at setting 7 or until the particles are completely suspended PrepSEQ9 Binding 1 Add 30 mL of 100 isopropanol to the Binding Solution bottle Solution 2 Label the bottle to indicate that it contains isopropanol then store the bottle at ambient temp
19. e 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 processing the sample 4 Add the following to the appropriate wells of the 96 deep well Lysis plate 100 uL of sample e Recommended Negative extraction NEG and extraction recovery controls ERC prepared according to the procedure on page 12 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 test samples if necessary on page 12 Alternatively make dilutions of the extracted DNA before running the PCR reaction 5 Centrifuge the Lysis plate at 1000 rpm for 1 minute in a benchtop centrifuge to spin down the solution on the wall 6 Add 10 uL of 5 M NaCl per 100 uL of sample 7 Add 70 uL of the Proteinase K and Proteinase K Buffer mix Centrifuge the plate at 1000 rpm for 1 minute in a benchtop centrifuge to spin down the solution on the wall 8 Select the program labeled PrepSEQ_ResDNA_2011 from the MagMax Express 96 9 Load the plates into the instrument in the order listed below After loading each plate press START to move the turntable a Comb loading plate b Elution plate with 200 uL of elution buffer c Wash 2 plate with 300 uL of wash buffer d Wash 1 plate with 300 uL of wash buffer e Lysis plate 10 Press START to be
20. emove ethanol from the Wash Solution Once dried the DNA will stay bound to the magnetic beads Do not over dry over dried beads are not easily resuspended Add 50 100 uL of Elution Buffer to each tube PrepSEQ Residual DNA Sample Preparation Kit User Guide 15 Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Automated protocol for residual DNA extraction 2 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 3 Centrifuge the tubes in a microcentrifuge at top speed for 15 seconds place the tubes in the magnetic stand and let the tubes stand for 2 minutes Transfer the liquid phase containing the eluted DNA to a new nonstick 1 5 mL microfuge tube Note The Magnetic Particles are magnetically captured in approximately 1 minute DNA is in the eluate 4 Centrifuge the tube at top speed gt 15 000 x g for 3 minutes to pellet the residual Magnetic Particles then place the tubes in the magnetic stand for 1 minute 5 Transfer the liquid phase containing the eluted DNA to the nonstick 1 5 mL microcfuge tube without disturbing the Magnetic 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 User Guide Pub no 4469836 to set up PCR for DNA quantitation
21. erature PrepS EQ Wash 1 Add 74 mL of 95 ethanol to the bottle that is labeled PrepSEQ Wash Solution Buffer Concentrate Concentrate then mix completely 2 Label the bottle to indicate that it contains ethanol then store the bottle at ambient temperature Proteinase K e Prepare a fresh mix that contains Proteinase K and Proteinase K Buffer for the Proteinase K total number of samples to be processed Prepare 70 uL of the fresh mix per Buffer mix 100 uL of sample e Include a 10 overage to account for pipetting losses PrepSEQ Residual DNA Sample Preparation Kit User Guide 11 Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Dilute test samples if necessary Lysis Solution Mix of Lysis Buffer tRNA and glycogen Comnoneni 1 reaction P per 100 pL of sample Proteinase K 10 uL Proteinase K buffer 60 UL Prepare a fresh mixture immediately before starting sample processing or during Proteinase K incubation Prepare 360 uL of the mix for sample preparation per 100 uL of sample Reagent Volume uL for 20 extractions Glycogen 5 mg mL 180 uL tRNA 10 mg mL 4 UL Lysis buffer 7600 UL Total 7784 uL Dilute test samples if necessary 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 before PrepSEQ Residual DN
22. f necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the 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 WARNING HAZARDOUS WASTE from instruments Waste produced by the instrument is potentially hazardous Follow the guidelines noted in the preceding General Chemical Handling warning WARNING 4L Reagent and Waste Bottle Safety 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 22 PrepSEQ Residual DNA Sample Preparation Kit User Guide Appendix B Safety Biological hazard safety Specific chemical handling CAS Chemical Phrase 26628 22 8 Sodium Azide Sodium azide may react with lead and copper plumbing to form highly explosive metal azides 50 01 1 Guanidine HCL Contact with acids or bleach liberates toxic gases DO NOT ADD acids or bleach to any liquid wastes containing this product 593 84 0 Guanidine Isothiocyanate Contact with acids or bleach liberates toxic gases DO NOT ADD acids or bleach to any liquid wastes containing this product Biological hazard safety
23. from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining support For the latest services and support information for all locations go to www lifetechnologies com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs e Submit a question directly to Technical Support techsupport lifetech com Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches PrepSEQ Residual DNA Sample Preparation Kit User Guide 25 Documentation and Support Limited product warranty Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 26 PrepSEQ Residual DNA Sample Preparation Kit User Guide For support visit lifetechnologies com support or email techsupport dlifetech com technologies lifetechnologies com 08 December 2
24. gin the PK digestion 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 When digestion is complete the instrument will pause and return the Lysis plate to the loading position 11 After the digestion step is complete add additional components to the Lysis plate a Remove the Lysis plate from the instrument b Add 360 uL of Lysis Solution using an 8 channel pipette see page 12 Pipet up and down two times to mix c Add 30 uL of Magnetic Particle suspension to the sample d Add 400 uL of Binding Solution and immediately pipet up and down three times to mix e Place the plate back into the instrument loading position then press START to begin binding PrepSEQ Residual DNA Sample Preparation Kit User Guide 17 Troubleshooting Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit 12 When DNA extraction is finished the instrument returns the Elution plate to the loading position 13 Use a multichannel pipette to carefully transfer 10 uL of eluate into the PCR reaction plate for the real time PCR assay Do not touch the particles Troubleshooting Observation Possible cause Action Poor extraction efficiency Overdrying Start the 5 minute timer before removing 300 uL low yields from the first 6 8 samples Then continue removing wash buffer from the remaining samples Magnetic Particles are attached too tightly to
25. icient of variation from this data SD Mean Quantity x 100 CV Based on this calculation you can then assign a CV value that indicates acceptable reproducibility 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 we recommend using the resDNASEQ Quantitative DNA kits as described in the resDNASEQ Quantitative DNA Kits User Guide Pub no 4469836 PrepSEQ Residual DNA Sample Preparation Kit User Guide 7 Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Kit contents and storage Kit contents and storage WARNING Read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Safety Data Sheets SDSs are available from www lifetechnologies com support The PrepSEQ Residual DNA Sample Preparation Kit Cat no 4413686 contains the PrepSEQ Nucleic Acid Extraction Kit Cat no 4400799 three boxes and the PrepSEQ Residual DNA Sample Preparation Kit Cat no 4399042 one box Kit components include Reagent Description Storage Cat no Box 1 PrepSEQ Nucleic Acid Extraction Kit 4400793 Lysis Buffer 2 bottles 50 mL bottle Store at room temperature 4400659 Binding Solution 1 empty bottle NA 4400789 Isopropanol Wash Buffer Concentrate 2 bottles 26 mL bottle St
26. lth Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en 9 WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards PrepSEQ Residual DNA Sample Preparation Kit User Guide 23 Appendix B Safety Biological hazard safety 24 PrepSEQ Residual DNA Sample Preparation Kit User Guide Documentation and Support Related documentation For information on new assays and updated 7500 product information go to www microseq com For brief instructions on using the PrepSEQ Residual DNA Sample Preparation Kit see the PrepSEQ Residual DNA Sample Preparation Kit Quick Reference Pub no 4469839 For information on performing PCR after sample extraction refer to the resDNASEQ Quantitative DNA Kits User Guide Pub no 4469836 For brief instructions on performing PCR after sample extraction refer to the resDNASEQ Quantitative DNA Kits Quick Reference Pub no 4469837 e For information on the MagMAX Express 96 DW instrument see the Applied Biosystems MagMAX Express 96 User Manual Pub no N07849 Portable document format PDF versions of this guide and the documents listed above are available at www lifetechnologies com Obtaining SDSs Safety Data Sheets SDSs are available
27. ne block heater is available after incubation reset it to 70 C for the elution step Note For samples with high protein concentration prolonging the PK digestion to 60 minutes can increase recovery Cool samples to room temperature Add 360 uL of freshly made Lysis Solution Mix per 100 uL of starting sample see Prepare reagents on page 11 PrepSEQ Residual DNA Sample Preparation Kit User Guide Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Manual protocol for residual DNA extraction 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 1 Vortex the Magnetic Particles until resuspension is complete The appearance of the mixture should be homogenous Add 30 uL of the Magnetic Particles to the 100 or 200 uL of sample Add 400 uL of the Binding Solution per 100 uL of starting sample close the caps and immediately invert the tubes twice to mix then vortex the tubes in the vortex adaptor for 5 minutes at setting 7 Quick spin the tubes in a microcentrifuge for 15 seconds at top speed 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 Wash the DNA For aspiration of liquid supernat
28. ore at room temperature 4400783 Elution Buffer 1 bottle 25 mL Store at room temperature 4400784 Proteinase K PK Buffer 1 bottle 50 mL Store at room temperature 4400787 Box 2 PrepSEQ Nucleic Acid Extraction Kit 4400795 Magnetic Particles 2 tubes 1 5 mL tube Store at room temperature 4401405 Box 3 PrepSEQ Nucleic Acid Extraction Kit 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 20 C 4404627 8 PrepSEQ Residual DNA Sample Preparation Kit User Guide Chapter 1 PrepSEQ Residual DNA Sample Preparation Kit Required materials Automation instrument plastics and accessories Required materials WARNING Read the Safety Data Sheets SDSs and follow the handling instructions Wear appropriate protective eyewear clothing and gloves Safety Data Sheets SDSs are available from www lifetechnologies com support The MagMAX Express 96 DW instrument Cat no 4456933 accessories include Item Source or Cat no 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 AM1
29. r up to 1 week and use it for multiple assays Preventing PCR contamination PCR assays require special laboratory practices to avoid false positive amplifications The efficiency and high sensitivity of these assays can lead to amplification of a single DNA molecule PrepSEQ Residual DNA Sample Preparation Kit User Guide 19 Appendix A Good Laboratory Practices Plate layout suggestions When preparing samples for PCR amplification Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or used during sample preparation when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate work areas and dedicated equipment and supplies for Sample preparation PCR setup PCR amplification Analysis of PCR products Never bring amplified PCR products into the PCR setup area Open and close all sample tubes and reaction plates carefully Try not to splash or spray PCR samples Keep reactions and components capped as much as possible Use a positive displacement pipette or aerosol resistant pipette tips Clean lab benches and equipment after use with freshly diluted 10 bleach solution Avoiding false To avoid false positives due to cross contamination positives due to s Prepare and close all negative control and unknown sample tubes before cross pipetting the positive control contamination Do not open tubes after
30. s a blank sample processed with the PrepSEQ kit Use the NEG to test for cross contamination of DNA extraction reagents e Three extraction recovery controls ERC Use the ERC to assess the efficiency of DNA extraction recovery and quantitation from test samples Additionally you can use the ERC to verify assay and system performance Spike each of the triplicate reactions separately to ensure that the mixture is homogenous DNA and other components in the reaction may not mix homogenously if you spike standard DNA into a larger sample volume and aliquot for testing Note Adjust the amount of DNA control added to the sample for those test samples that contain higher background levels of DNA To ensure accurate results the amount of DNA control that you add to a test sample should be 2 10 times the amount of DNA measured in the test sample without the addition of the DNA control To calculate the efficiency of DNA recovery and quantitation from the test samples subtract the amount of DNA measured in the sample without the addition of DNA control from the amount of DNA measured in the ERC sample For each test sample 1 Prepare the negative extraction controls Label three 2 mL Safe Lock tubes as NEG Add 100 uL of PBS to each tube 2 Prepare the extraction recovery controls Label three 2 mL Safe Lock tubes as ERC lt n gt pg where lt n gt is the pg amount As an example this step describes the preparation of an E
31. tents About THIS GUIDE oca daa 5 REVISION HISTORY nsten aa aa A ie HH HNH e_ 5 RURDOS Or e arca o iaa add ae th do en a dd as e e T 5 Pr e e 5 Hi CHAPTER 1 PrepSEQ Residual DNA Sample Preparation Kit 7 Product description EDED D OJ jJj N NnN nE HnDnEnEnE EDED eee eee eee eect eae oe nek pease Pee pear Pele 7 Kiticontents and StOFAUe ear ta al tides ts ds 8 Required materials HHH ines Sap 9 Automation instrument plastics and accessories kk kk kk kk kK KK KK KK KK KK KK 9 Required materials not included in the kit WL kk kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK 10 Workflow for residual DNA extraction manual or automated from CHO E coli Human Vero Pichia NSOS and e A y q b raa A tarteb EE Ne CRs O n iat Ate A Ay 11 Prepare EGG GDS ac ia ea de de DD rrr e oe 11 Prepare magnetic beads M M W kk kk kk kk kk kk kK kk kk KK kK kK kk kk kk kk kk kk kk kk kk kk kk kk kk 11 PrepSEQ Binding Solution s Ak dk dakae kala k Kalek e n AET eka Ak A Wi Giga ese nee 11 PrepSEQ Wash Buffer Concentrate 11 Proteinase K Proteinase K Buffer Mix HW kk kk kk kk kk kK kk kK KK KK KK KK KK KK KK KK KK KK k 11 Lysis Solution Mix of Lysis Buffer tRNA and glycogen kk kk kk kk KK KK KK KK KK 12 Dilute test samples if necessary W WWW kk kk kk kk KK KK KK rr 12 Prepare triplicate reactions rr 12 Recommended Prepare the extraction controls 0c KK KK KK KK KK KK 13 G
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