Complete Protocol
Contents
1. ethanol to each full bottle of Wash Buffer WBA Mix well before use Note Do not add ethanol to Prepared Wash Buffer WBC from Cat A2751 This reagent contains ethanol and is ready for use Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM341 Revised 7 13 Page 5 Promega 4 B Manual DNA Purification Materials to Be Supplied by the User HSM 2 0 Instrument Cat A2715 with HSM 2 0 Tube Rack HSM 2 0 Tube Rack Stand and computer with HSM 2 0 Instrument software installed see HSM 2 0 Instrument Operating Manual TM389 for details e 47 5 50 ethanol at room temperature prepared using Molecular Biology erade water see Section 4 A e 50ml conical tubes one per sample e g Corning Cat 430291 e 10ml pipettes and pipette aid e Optional A repeater pipette Gilson or Eppendorf for adding reagents to larger numbers of samples Be sure to remove all lysate Prepared Wash Buffer and ethanol from the tubes in Steps 7 9 11 and 13 Failure to remove all lysate Prepared Wash Buffer and ethanol will result in decreased purity of the eluted DNA 1 Place the HSM 2 0 Tube Rack on the HSM 2 0 Tube Rack Stand 2 Place one empty uncapped 50ml conical tube per sample into the HSM 2 0 Tube Rack Add blood samples to the uncapped 50ml tubes Note The ReliaPrep Large Volume2. HT gDNA Isolation System is designed to process whole blood in uncapped tubes Do not dilute blood prior to processing 3 Transfer the HSM 2 0 Tube Rack to the HSM 2 0 Instrument 4 Launch the HSM 2 0 Instrument software and select the appropriate protocol for the desired sample type Enter the blood volume and the software will provide reagent volumes based on the volume provided If you are processing multiple blood volumes refer to Table 1 for reagent volumes Table 1 Reagent volumes ml across multiple sample volumes Sample Volume ml Reagent per sample 3G 4 5 6 7 8 9 10 Proteinase K 0 060 0 080 0 100 0 12 014 016 0 180 0 20 RNase Optional 0 060 0 080 0 100 0 12 0 140 016 0 180 0 20 Alkaline Protease 075050006251075 MkeviS IW LIZ 12 Lysis Buffer 3 00 4 00 5 00 6 00 7 00 800 9 00 10 00 Binding Buffer Saal ahel GUO 70 roel Shei ING etn 2180 Prepared Wash Buffer 5 00 5 60 610 670 7 30 790 840 9 00 50 Ethanol 400 460 510 5 70 630 690 740 8 00 Note that samples with volumes less than 3ml are processed using the reagent volumes for 3ml of whole blood Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM341 Printed in USA Page 6 Revised 7 13 Promega 5 Add the volume of Proteinase K Solution displayed onscreen to each tube 6 Optional Add the indicated volume of RNase A
3. a registered trademark of Microsoft Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are subject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM341 Printed in USA Page 12 Revised 7 13 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 608 274 4330 Fax 608 277 2516 www promega com TM341 Revised 7 13
4. indicated on screen to each tube 7 Select OK to start shaking samples at 500rpm for 1 minute After the beep add the volume of Alkaline Protease indicated on screen to each tube Select OK to start shaking samples at 500rpm for 1 minute 8 Add the appropriate volume of Lysis Buffer to each sample After Lysis Buffer addition the HSM 2 0 Instrument incubates the samples at 65 C for 30 minutes with shaking at 500rpm followed by 10 minutes of shaking at 500rpm without heat 9 Add the appropriate volume of Binding Buffer to each sample Select OK to start shaking samples at 550rpm for 3 minutes After shaking thoroughly resuspend the ReliaPrep Resin and add the appropriate volume of resin to each sample as shown on screen Select OK to start shaking samples at 550rpm for 20 minutes Thoroughly resuspend the ReliaPrep Resin before dispensing 10 After the shaker stops the HSM 2 0 Instrument collects the ReliaPrep Resin at the side of the tube for 20 minutes When prompted by the instrument slowly aspirate and dispose of the lysate in the tube O Remove all lysate from the tubes before adding wash buffer in Step 11 After the initial removal perform an additional aspiration to ensure that all lysate is removed Note Rapid aspiration of the lysate may result in loss of the resin 11 Add the amount of Prepared Wash Buffer indicated on screen to each tube Press Enter to start samples sh
5. minimized DNA degradation 7 References 1 Wilfinger W Mackey K and Chomczynski P 1997 Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity BioTechniques 22 474 81 2 Glasel J A 1995 Validity of nucleic acid purities monitored by 260nm 280nm absorbance ratios BioTechniques 18 62 3 3 Manchester K L 1995 Value of Ay o A2g9 ratios for measurement of purity of nucleic acids BioTechniques 19 208 10 8 Related Products Product Size Cat HSM 2 0 Instrument 1 each A2715 HSM 2 0 Tube Rack 1 each A2713 HSM 2 0 Tube Rack Stand 1 each A2714 2 2ml Square Well Deep Well Plate 50 case V6781 RNase A Solution 5ml A7974 20X TE Buffer pH 7 5 25ml A2651 Alkaline Protease APA 130ml A1721 Cell Lysis Buffer CLD 1 400ml A1731 Binding Buffer BBA 1 600ml A1741 ReliaPrep Resin 115ml A1752 Prepared Wash Buffer WBC 3 000ml A2681 Proteinase K PK Solution 23ml A5051 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM341 Revised 7 13 Page 11 Promega 8 Related Products continued Product Size Cat Nuclease Free Water 500ml P1197 1 000ml P1199 Tissue Lysis Buffer TLA 500ml A5091 Bottle for 50 Ethanol each A2691 2010 2013 Promega Corporation All Rights Reserved ReliaPrep is a trademark of Promega Corporation Windows is
6. 30nm 260nm and 280nm before reporting these values and ratios Before performing this calculation check with your spectrophotometer user manual to determine whether your instrument performs this calculation 6 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Symptoms Causes and Comments Low DNA yield Insufficient mixing during elution During successful elution the ReliaPrep Resin is thoroughly resuspended during the mixing step and the elution solution is a homogeneous dull brown color During poor elution the ReliaPrep Resin remains in a loose clump swirling at the bottom of the tube during the shaking steps Be sure that the ReliaPrep Resin is thoroughly resuspended during the elution step Failure to resuspend the ReliaPrep Resin adequately before use Thoroughly resuspend ReliaPrep Resin in the reagent bottle before dispensing for sample purification Sample contained too few white cells per 1ml of blood DNA yield depends on the amount of starting material Blood samples with low white cell counts will have reduced yields due to low Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM341 Revised 7 13 Page 9 Promega 6 Tr
7. TECHNICAL MANUAL ReliaPrep Large Volume HT gDNA Isolation System Instructions for use of Products A1751 AND A2751 Pig Promega ReliaPrep Large Volume HT gDNA Isolation System All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail techserv promega com Des DVO SCRUTINY a E E EEE E 1 2 Product Components and Storage Conditions 0 0 ee eeeeteeeeeeeneees 3 3 General Considerations iia cts ccacisassxcessteicscotesccvanseysesstatessincvsocesieccossscteessnancester s 4 A Comparison with Organic and Precipitation Based Methods 4 B Sample Processina Gor all aig tami epentennT meen Teer erreNenT Teeter nt tT aai 4 C 2 0h 0 eer a E etree ee E eee 4 BE Cn on kerin oul e E S 4 E Manual Versus Automated Processing ssessseeirisessssssrrirresssserrrrireess 5 4 Genomic DNA Purification Procedure 0 cecescesesssecsestesteceeseeseeneeneeseens 9 O A eal EOI A E E T A 5 B Manual DNA PUN CA Olosan E seers serene 6 5 Quantitation and Analysis of Isolated Genomic DNA ececeeeeees 9 G TOONS NOC roina E E RE 9 A EIN E A E E A T e 11 Bh Related ROC seep ee 11 1 Description The ReliaPrep Large Volume HT gDNA Isolation System isolates genomic DNA gDNA f
8. aking at 500rpm for 2 minutes The ReliaPrep Resin should dislodge from the side of the tube during this shaking step 12 After shaking for 2 minutes the HSM 2 0 Instrument prompts you to mix the samples Pipet the samples at least 10 times to thoroughly disperse the resin Following this the instrument will shake again for 2 minutes to wash the resin 13 The HSM 2 0 Instrument collects the ReliaPrep Resin for 3 minutes Slowly aspirate the fluid from each tube when prompted by the instrument Remove all wash buffer before adding fresh Prepared Wash Buffer in Step 14 After the initial removal perform an additional aspiration to ensure that all Wash Buffer is removed 14 Add the amount of Prepared Wash Buffer indicated on screen to each tube Select OK to start samples shaking at 500rpm for 3 minutes then shaking for 3 minutes at 700rpm Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM341 Revised 7 13 Page 7 Promega 4 B Manual DNA Purification continued 15 The shaker will stop and the HSM 2 0 Instrument collects the ReliaPrep Resin at the side of the tube for 3 minutes Slowly aspirate the fluid from each tube when prompted by the instrument Remove all Wash Buffer before adding ethanol in Step 16 After the initial removal perform an additional aspiration to
9. ed at each step This ensures optimal purification of genomic DNA away from sample contaminants Incomplete lysis of blood cells Failure to add sufficient volume of Proteinase K Solution Alkaline Protease or Cell Lysis Buffer will result in incomplete lysis and digestion of cells and proteins ReliaPrep Resin in final eluate Concentrated DNA solutions can be viscous Additional time may be required to capture the ReliaPrep Resin from viscous solutions Small amounts of resin may be transferred during manual purification The eluted DNA should be collected slowly and any residual resin removed from the eluted DNA by performing additional magnetic captures or a centrifugation Madison WI 53711 5399 USA Fax 608 277 2516 www promega com Printed in USA Revised 7 13 Promega Symptoms Causes and Comments Degraded DNA Nucleases were introduced during purification and handling Use nuclease free plasticware or glassware Use aerosol resistant tips during all pipetting steps Wear gloves at all times Nucleases introduced after elution will degrade DNA Add 10X or 20X TE buffer to a final concentration of 1X in the final eluate to protect eluted DNA from nucleases DNA was degraded before the purification process If sample DNA was degraded before purification the resulting purified DNA also will be degraded Repeat DNA purification with new starting material Be sure that starting material has been handled to
10. ensure that all Wash Buffer is removed 16 Add the amount of 47 5 50 ethanol shown on screen to each sample Select OK to start samples shaking samples at 500rpm for 4 minutes 17 The shaker will stop and the HSM 2 0 Instrument collects the ReliaPrep Resin at the side of the tube for 3 minutes Slowly aspirate the fluid from each tube when prompted by the instrument Remove all ethanol from the tubes before adding Nuclease Free Water in Step 18 After the initial removal perform an additional aspiration to ensure that all ethanol is removed 18 Add 1 5ml of Nuclease Free Water to each tube The HSM 2 0 Instrument first disperses the resin in the Nuclease Free Water by shaking for 3 minutes at 600rpm and then incubates the samples at 70 C for 20 minutes with shaking at 400rpm Note A volume of 1 5ml of Nuclease Free Water will produce a final eluted volume of approximately 1 3ml The final recovered volume will be approximately 200ul less than the volume of Nuclease Free Water added Adding less Nuclease Free Water will reduce the total eluted volume creating eluates of higher concentration but may decrease total yields Do not elute with less than 700ul or more than 1 7ml of Nuclease Free Water 19 The HSM 2 0 Instrument collects the ReliaPrep Resin at the side of each tube for 5 minutes Transfer each eluate to a 1 5ml tube or 96 well plate Note Removing all ReliaPrep Resin from the eluate requires a centrifuga
11. ing system with methods to process 1 10ml of blood These methods guide the user through the purification process with specific instructions on reagent addition and waste removal Solutions are typically added with a pipette and aspirated using a vacuum aspirator Automated Processing Automated processing is available using a liquid handling workstation and integrated HSM 2 0 Instrument Automated processing provides scalability to adapt easily to daily needs the instrument senses the sample volume in each tube and scales reagent volumes appropriately without user intervention The method will process variable sample volumes within a single run as well as variable batch sizes between runs For more information about liquid handling options contact Promega at techserv promega com 4 Genomic DNA Purification Procedure This overview describes the liquid handling and purification steps required to isolate genomic DNA from whole blood using the ReliaPrep Large Volume HT gDNA Isolation System This protocol can be performed manually using the preloaded methods in the HSM 2 0 Software Section 4 B or an automated liquid handling workstation 4 A Reagent Preparation 47 5 50 ethanol Combine equal volumes of 95 100 USP ACS or Molecular Biology grade ethanol with Molecular Biology grade water Mix Note Use of ethanol that contains methanol or isopropanol may cause decreased DNA yield and purity Prepared WBA Add 150ml of the 47 5 50
12. olation System A2751 Each system contains sufficient reagents for 96 isolations from up to 10ml each or 320 x 1 3ml in a scalable format Includes 23ml Proteinase K PK Solution e 130ml Alkaline Protease APA e 1400m Cell Lysis Buffer CLD e 1 600ml Binding Buffer BBA e 115ml ReliaPrep Resin e 3 000ml Prepared Wash Buffer WBC e 4x150m Nuclease Free Water Storage Conditions Store the ReliaPrep Large Volume HT gDNA Isolation System at room temperature 15 30 C Do not refrigerate or freeze any of the reagents Naming Conventions Throughout this Technical Manual the solutions supplied with the ReliaPrep Large Volume HT gDNA Isolation System are referred to as follows Proteinase K PK Solution as Proteinase K Solution Alkaline Protease APA as Alkaline Protease Cell Lysis Buffer CLD as Cell Lysis Buffer Binding Buffer BBA as Binding Buffer Wash Buffer WBA as Wash Buffer and Prepared Wash Buffer WBC or Wash Buffer WBA with added ethanol as Prepared Wash Buffer 2800 Woods Phone 608 274 4330 Hollow Road Madison WI 53711 5399 USA Fax 608 277 2516 www promega com Part TM341 Page 3 Promega Corporation Toll Free in USA 800 356 9526 Printed in USA Revised 7 13 Promega 3 General Considerations 3 A Comparison with Organic and Precipitation Based Methods The ReliaPrep Large Volume HT gDNA Isolation System addresses the challenges of traditional genomic DNA pu
13. oubleshooting continued Symptoms Low DNA yield continued Eluted DNA is discolored Promega Corporation 2800 Woods Toll Free in USA 800 356 9526 Part TM341 Page 10 Hollow Road Phone 608 274 4330 Causes and Comments Sample size exceeded the processing capacity of the system The ReliaPrep Large Volume HT gDNA Isolation System is optimized to purify DNA from 1 10ml of whole blood Processing blood samples that are outside of this range will result in reduced DNA yield and concentration Exceeding the 10ml limit will cause the required processing volumes to exceed the maximum volume of a 50ml tube The ReliaPrep Large Volume HT gDNA Isolation System is optimized for whole blood samples that contain between 4 5 x 10 and 1 x 10 white cells ml Whole blood samples containing more white cells per 1ml may cause slower capture of the ReliaPrep Resin during the purification process resulting in reduced yield Elution volume was too low Using a smaller elution volume will increase purified DNA concentration but also will decrease total yield The final eluted volume needs to be at least 1ml for the ReliaPrep Resin to be captured by the magnets within the HSM 2 0 Instrument Successful use of the ReliaPrep Large Volume HT gDNA Isolation System depends on use of the correct reagents in the correct order Check to ensure that all protocol steps were followed correctly and that the correct reagents were us
14. rification methods that rely on centrifugation based organic or precipitation chemistries by isolating genomic DNA from the entire lysed whole blood sample The ReliaPrep Large Volume HT gDNA Isolation System efficiently purifies genomic DNA regardless of whether blood samples are fresh or frozen 3 B Sample Processing Capacity Genomic DNA yield depends on 1 volume of whole blood processed and 2 number of white blood cells ml The ReliaPrep Large Volume HT gDNA Isolation System purifies genomic DNA from up to 10ml of whole blood in one purification procedure This volume limit assumes that the average number of white blood cells per 1ml of whole blood from a normal healthy adult is between 4 5 x 106 and 1 1 x 107 cells ml Therefore the cell number limit of the ReliaPrep Large Volume HT gDNA Isolation System is approximately 1 x 10 leukocytes Exceeding the recommended volume or cell number may result in clumping of the ReliaPrep Resin and reduced gDNA yield and quality 3 C Elution The recommended elution volumes in the ReliaPrep Large Volume HT gDNA Isolation System protocol may be adapted to accommodate downstream applications that require a specific DNA concentration The recommended elution volume range is 700 1 700u1 The final recovered volume will be approximately 200ul less than the volume of Nuclease Free Water added for a final elution volume of 500 1 500u1 Eluting DNA ina smaller volume will increase the pu
15. rified DNA concentration but also will result in lower total yield In addition elution volumes of less than 1ml may result in poor resuspension of the ReliaPrep Resin causing decreases in performance and increased resin carryover into eluates For optimal elution of genomic DNA from the ReliaPrep Resin add room temperature Nuclease Free Water to the ReliaPrep Resin mix and heat with the HSM 2 0 Instrument Failure to heat after adding the Nuclease Free Water will decrease DNA yield and concentration For optimal elution the ReliaPrep Resin must be heated and mixed vigorously during mix steps to efficiently release genomic DNA 3 D Additional Sample Types The ReliaPrep Large Volume HT gDNA Isolation System has been adapted to isolate gDNA from additional sample types including blood fractions and saliva For a complete list of compatible sample types and protocols visit www promega com products biobanking Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM341 Printed in USA Page 4 Revised 7 13 Promega 3 E Manual Versus Automated Processing For more information about the HSM 2 0 Instrument see the HSM 2 0 Instrument Technical Manual TM389 at www promega com protocols Manual Processing The HSM 2 0 Instrument is controlled by software installed on a computer with the Windows 7 operat
16. rom blood samples ranging from 1ml to 10ml of blood in a scalable format The chemistry eliminates tedious centrifugation steps as well as the use of hazardous chemicals which are inherent in precipitation based chemistries The system has been automated on liquid handling workstations allowing walkaway purification of genomic DNA from 1 10ml of whole blood regardless of sample storage or shipping conditions The system is scalable and uses only the amount of reagents required to process each sample based on sample volume maximizing efficiency and value per prep Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM341 Revised 7 13 Page 1 Promega 1 Description continued Sample processing is simplified through the use of the HSM 2 0 Instrument Cat A2715 which heats shakes and magnetizes sample tubes in one position throughout the extraction process from lysis to elution This system removes resource limitations from sample processing and increases automated method robustness and reliability Genomic DNA yields from normal 10ml whole blood samples are typically 200 4002 depending on white blood cell count in a final eluted volume of 1ml Recovered DNA exhibits good purity with A260 A250 ratios greater than 1 7 and Ax 9 Azo ratios between 1 8 and 2 2 The ReliaPrep Large Volume HT gDNA Isola
17. tion System in combination with the HSM 2 0 Instrument offers a dependable and efficient option for automated isolation of high quality DNA from large volume blood samples Hardware Requirements For manual DNA purification from 1 32 blood samples 1 10ml each the hardware listed below is required Product Quantity Cat HSM 2 0 Instrument processes up to 32 samples per instrument 1 A2715 1The HSM 2 0 Instrument is supplied with one HSM 2 0 Tube Rack and one HSM 2 0 Tube Rack Stand For people who wish to purchase additional HSM 2 0 Tube Racks and HSM 2 0 Tube Rack Stands these items are available separately Available Separately Cat HSM 2 0 Tube Rack A2713 HSM 2 0 Tube Rack Stand A2714 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM341 Printed in USA Page 2 Revised 7 13 Promega 2 Product Components and Storage Conditions Product Cat ReliaPrep Large Volume HT eDNA Isolation System A1751 Each system contains sufficient reagents for 96 isolations from up to 10ml each or 320 x 1 3ml in a scalable format Includes 23ml Proteinase K PK Solution e 130ml Alkaline Protease APA e 1 020ml Cell Lysis Buffer CLD 2x 765ml Binding Buffer BBA e 115ml ReliaPrep Resin e 3x 815ml Wash Buffer WBA e 2x150ml Nuclease Free Water Product Cat ReliaPrep Large Volume HT eDNA Is
18. tion step 2 minutes at maximum speed in a microcentrifuge if eluates are in tubes or 10 minutes at 2 500 x g if eluates are in a 96 well plate We recommend adding concentrated TE buffer 10X or 20X to eluted samples to a final concentration of 1X TE buffer for long term storage Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM341 Printed in USA Page 8 Revised 7 13 Promega 5 Quantitation and Analysis of Isolated Genomic DNA DNA quality and concentration can be determined by a variety of means including spectrophotometry dye fluorescence gel electrophoresis and quantitative PCR Different DNA quantitation methods frequently return dissimilar absolute quantitation values We recommend that users select analysis methods for DNA quality and quantity that best predict success for the intended downstream applications A260 A259 ratio can be used to determine DNA purity with a number of important limitations 1 3 An Axgo Ago ratio between 1 7 and 2 0 is generally accepted as representative of a high quality DNA sample The ratio can be calculated after subtracting the non nucleic acid absorbance at A399 Az reading Asx reading DNA purity A2s0 Azs Azs reading Asx reading Note Many spectrophotometers automatically subtract the absorbance at a reference value around 340nm from the absorbance at 2
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