User Guide
Contents
1. for PCR strips skirted non skirted or half skirted PCR plates Agencourt SPRIStand 6 Position Tube Magnet Beckman Coulter Genomics Cat A29182 for 1 5 mL 1 7 mL and 2 0 mL microcentrifuge tubes Invitrogen DynaMag 96 Side Invitrogen Cat 123 31D for PCR strips non skirted or half skirted PCR plates Invitrogen DynaMag 96 Side Skirted Invitrogen Cat 120 27 for skirted PCR plates Promega MagnaBot I Magnetic Separation Device Promega Cat V8351 for PCR plates Disposable gloves Kimwipes Ice bucket Cleaning solutions such as DNA OFF MP Biomedicals Cat QD0500 To Order Affymetrix www affymetrix com Agilent www agilent com Beckman Coulter Genomics www beckmangenomics com Covaris www covarisinc com Invitrogen Life Technologies www invitrogen com MP Biomedicals www mpbio com Nanodrop www nanodrop com Sigma Aldrich Inc www sigmaaldrich com 9 Ill Planning the Experiment Ovation SP Ultralow Library Systems A Input DNA Requirements The Ovation SP Ultralow Library Systems are designed to work with 1 to 100 ng of fragmented genomic dsDNA or ds cDNA DNA samples must be free of contaminating proteins RNA organic solvents including phenol and ethanol and salts Use of a com mercially available system for DNA cDNA isolation is recommended The A260 A280 ratio for DNA samples should be in excess of 1 8 Use of DNA samples with lower ratios may resu2. A N A S01588 Bead Binding Solution 2of2 N A N A S501589 Bead Wash Solution 2of2 N A N A S01590 Elution Buffer 2of2 N A N A P01198 a 2 of 2 N A N A soia Mifeaton Beads2 sepratay NA ua B Additional Equipment Reagents and Labware Required Materials e Equipment Mondrian SP Workstation NuGEN Part No 8000 or Mondrian SP Workstation NuGEN Part No 8100 Covaris S series Sonication System to fragment input DNA Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic analysis of nucleic acids Microcentrifuge for individual 1 5 mL and 0 5 mL tubes 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer Thermal cycler with 0 2 mL tube heat block heated lid and 100 pL reaction capacity for library enrichment Nanodrop UV Vis Spectrophotometer or appropriate spectrophotometer and cuvettes for quantification of fragmented DNA 8 Components Ovation SP Ultralow Library Systems Reagents Ethanol Sigma Aldrich Cat E7023 for purification steps 1X TE buffer low EDTA pH 8 0 Affymetrix Cat 75793 Supplies and Labware Nuclease free pipette tips 1 5 mL and 0 5 mL RNase free microcentrifuge tubes 0 2 mL individual thin wall PCR tubes 8 X 0 2 mL strip PCR tubes or 0 2 mL thin wall PCR plates Magnetic separation device options Agencourt SPRIPlate Ring Super Magnet Plate Beckman Coulter Genomics Cat 432782
3. Components and Reagents Part No 8033 eas 8033 8033 8033 a NUMBER DESCRIPTION BOX VIAL CAP NUMBER S01627 End Repair Buffer Mix 1 of 2 Blue ER1 ver 5 01510 End Repair Enzyme Mix 1 of 2 Blue ER2 ver 4 S01626 End Repair Enhancer 1 of 2 Blue ER3 01625 End Repair Enhancer Buffer Mix 1 of 2 Blue ER4 01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 S01669 DR Multiplex Ligation Adaptors 1 of 2 Yellow L2V9DR BC1 01670 1 8 L2V9DR BC2 S01671 L2V9DR BC3 01672 L2V9DR BC4 S 01673 L2V9DR BC5 S01674 L2V9DR BC6 S 01675 L2V9DR BC7 S01676 L2V9DR BC8 01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 S 01665 Amplification Buffer Mix 1 of 2 Red P1 vER 2 S01607 Amplification Primer Mix 1 of 2 Red P2 ver 5 S01667 Amplification Enzyme Mix 1of2 Red P3 S01668 DMSO 1of2 Red P4 S01001 Nuclease free Water 1 of 2 Green D1 PO1190 Mondrian SP Cartridges x4 2 of 2 N A N A S01561 Cartridge Filler Fluid x4 2of2 N A N A S01556 Sample Concentration Solution 2of2 N A N A S01588 Bead Binding Solution 2of2 N A N A S01589 Bead Wash Solution 2of2 N A N A 5 Ovation SP Ultralow Library Systems 6 Components Ovation SP Ultralow Library Systems Ovation SP Ultralow DR Multiplex System 1 8 Components and Reagents Part No 8033 continued oe 8033 8033 8033 oa NUMBER DESCRIPTION BOX VIAL CAP NUMBER S 01590 Elution Buffer 2 of 2 N A N A 01198 Ovation SP
4. Ultralow Cartridge 2of2 N A N A Loading Guide x4 501307 Pen aie RNAClean XP Shipped N A N A Purification Beads x2 separately Table 2 Ovation SP Ultralow DR Multiplex System 9 16 Components and Reagents Part No 8034 ee 8034 8034 8034 a NUMBER DESCRIPTION BOX VIAL CAP NUMBER S01627 End Repair Buffer Mix 1of2 Blue ER1 ver 5 01510 End Repair Enzyme Mix 1 of 2 Blue ER2 ver 4 01626 End Repair Enhancer 1 of 2 Blue ER3 01625 End Repair Enhancer Buffer Mix 1 of 2 Blue ER4 01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 S01677 DR Multiplex Ligation Adaptors 1 of 2 Yellow L2V9DR BC9 01678 9 16 L2V9DR BC10 01679 L2V9DR BC11 01680 L2V9DR BC12 01681 L2V9DR BC13 01682 L2V9DR BC14 01683 L2V9DR BC15 01684 L2V9DR BC16 01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 S01665 Amplification Buffer Mix 1 of 2 Red P1 ver 2 S 01607 Amplification Primer Mix 1 of 2 Red P2 ver 5 S01667 Amplification Enzyme Mix 1of2 Red P3 7 Components Ovation SP Ultralow Library Systems Ovation Ultralow DR Multiplex System 9 16 Components and Reagents Part No 8034 continued 8034 8034 PART DESCRIPTION Box viaccap VAL NUMBER NUMBER S01668 DMSO 1 of 2 Red P4 01001 Nuclease free Water 1 of 2 Green D1 PO1190 Mondrian SP Cartridges x4 2 of 2 N A N A S01561 Cartridge Filler Fluid x4 2of2 N A N A S01556 Sample Concentration Solution 2of2 N
5. above e Mondrian SP Cartridge failed or Mondrian SP cartridge status is undetermined do not use the cartridge and contact NUGEN Technical Support IV Protocol 15 Ovation SP Ultralow Library Systems F Protocol for the Ovation SP Ultralow Library Systems on the Mondrian SP Cartridge 1 Sample Solution Preparation Prepare sample solution for loading onto the cartridge this is done on a per sample basis and not as a master mix You must prepare and process no fewer than eight samples on each cartridge Table 3 Sample Solution Preparation COMPONENT VOLUME 1 to 100 ng sheared dsDNA in water or low EDTA TE Variable up to 23 5 uL Agencourt RNAClean XP beads 4 0 uL Sample Concentration Solution 27 5 uL Nuclease free water D1 to 55 uL final Variable volume Total volume 55 pL Ensure Agencourt RNAClean XP beads are at room temperature and completely resus pended prior to use Mix each sample solution well and incubate at room tempera ture approximately 23 C for 10 minutes The above recipe is meant for one sample Prepare sample solution for each sample to be processed 2 Ovation SP Ultralow Reagent Master Mix Preparation Prepare End Repair Master Mix 1 Thaw End Repair Buffer Mix ER1 ver 5 at room temperature vortex to mix well and spin down briefly Keep End Repair Enzyme Mix ER2 ver 4 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube
6. loss of beads during the ethanol washes will impact DNA yields so make certain to minimize bead loss throughout the procedure e Ensure that the ethanol wash is freshly prepared from fresh ethanol stocks at the indicated concentration Lower percent ethanol mixes will reduce recovery e During the ethanol washes keep the samples on the magnet The beads should not be allowed to disperse the magnet will keep the beads on the walls of sample wells or tubes in a small ring It is critical that all residual ethanol be removed prior to continuing with the next step Therefore when removing the final ethanol wash first remove most of the ethanol then allow the excess to collect at the bottom of the tube before removing the remaining ethanol This reduces the required bead air drying time e After drying the beads for the time specified in the protocol inspect each tube carefully and make certain that all the ethanol has evaporated before proceeding e It is strongly recommended that strip tubes or partial plates are firmly placed when used with the magnetic plate We don t advise the use of individual tubes as they are difficult to position stably on the magnetic plates D DNA Fragmentation Use a Covaris S Series System to fragment your source double stranded gDNA or cDNA to the desired length following the manufacturer s recommendations E Cartridge Quality Control Check The Mondrian SP Cartridge QC protocol is a QC check that we recomm
7. will impact the amount of DNA recov ered so ensure beads are not removed with the binding buffer or the wash With the plate still on the magnet add 200 uL of freshly prepared 70 ethanol to each sample well and allow plate to stand for 30 seconds Remove the 70 ethanol wash using a pipette Repeat the 70 ethanol wash 1 more time for a total of 2 washes Note With the final wash it is critical to remove as much of the ethanol as pos sible Use 2 pipetting steps and allow excess ethanol to collect at the bottom of the tubes after removing most of the ethanol in the first pipetting step VI Enriched Library Purification 24 Ovation SP Ultralow Library Systems 12 13 14 1 16 17 Air dry the beads on the magnet for approximately 2 minutes Inspect each tube carefully to ensure that all the ethanol has evaporated It is critical that all residual ethanol be removed prior to continuing Remove tubes from magnet Add 33 uL 1X TE to the dried beads Mix thoroughly to ensure all the beads are resuspended Transfer tubes to magnet and let stand for 2 minutes Carefully remove 30 uL of the eluate ensuring as few beads as possible are carried over and transfer to a fresh set of tubes When pipetting any portion of this eluted library downstream be sure to use a magnet stand to minimize bead carryover into any ensuing reactions Proceed to Step C Quantitative and Qualitative Assessment of the Purified Enriched
8. 1001 Vil 26 Technical Support Ovation SP Ultralow Library Systems For help with any of our products please contact NUGEN Technical Support at 650 590 3674 direct or 888 654 6544 option 2 toll free US only You may also send faxes to 888 296 6544 toll free or email techserv nugeninc com In Europe contact NUGEN at 31 0 135780215 Phone or 31 0 135780216 Fax or email europe nugeninc com In all other locations contact your NUGEN distributor for technical support VIII Appendix A Sequences of the DR Barcodes in the Multiplexed Reactions Table 8 Barcode sequences for dedicated read DR adaptors LIGATION ADAPTOR MIX BARCODE SEQUENCE L2V9DR BC1 AAGGGA L2V9DR BC2 CCTTCA L2V9DR BC3 GGACCC L2V9DR BC4 TTCAGC L2V9DR BC5 AAGACG L2V9DR BC6 CCTCGG L2V9DR BC7 GGATGT L2V9DR BC8 TTCGCT L2V9DR BC9 ACACGA L2V9DR BC10 CACACA L2V9DR BC11 GTGTTA L2V9DR BC12 TGTGAA L2V9DR BC13 ACAAAC L2V9DR BC14 CACCTC L2V9DR BC15 GTGGCC L2V9DR BC16 TGTTGC 27 Ovation SP Ultralow Library Systems VIII Appendix 28 Ovation SP Ultralow Library Systems B Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 What kind of sequencing primers can use with your library The Ovation SP Ultralow Library Systems are designed for use with the standard Illumina sequencing primers for both single end and paired end sequencing app
9. Libraries Quantitative and Qualitative Assessment of the Purified Enriched Libraries Run the samples on the Bioanalyzer DNA Chip 1000 The fragment distribution for 200 bp inserts is shown in Figure 6 note that the actual size of the libraries appears closer to 300 bp due to the additional length conferred by the adap tors that have been ligated onto the initial 200 bp fragment The fragment distribution is dependent on the initial fragment size Adaptor dimer formation is effectively eliminated if the protocol is followed Any deviation from the protocol may result in dimer formation which may appear as low molecular weight spikes approximately 120 bp on a Bioanalyzer trace VI Enriched Library Purification 25 Ovation SP Ultralow Library Systems Figure 6 The Bioanalyzer traces represent 8 with input amounts ranging from 0 1 to 10 ng of dsDNA lanes from the Mondrian SP Cartridge The total DNA recov ered from the cartridge was subjected to 18 cycles of PCR for enrichment and equal volumes were applied to the Bioanalyzer DNA 1000 chip 300 Sample 1 Sample 5 250 Sample2 Sample 6 Sample3 Sample 7 Sample 4 N Q O a Q O Fluorescence Units a oO T i T T T T T i T T 15 50 100 150 200 300 400 500 700 1500 Size bp 2 Validate the library as described in Illumina user guides for DNA library construc tion e g Genomic DNA Sample Prep Manual Cat FC 102
10. Ovation SP Ultralow Library Systems PART NOS 8033 8034 ga NUGEN imagine more from lesse Patents Licensing and Trademarks 2012 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugeninc com NUGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NUGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners Specific information on patents trademarks and licenses related to the Mondrian SP Universal Cartridge the Mondrian SP Cartridge the Mondrian SP Workstation and the Mondrian SP Workstation may be found in the Mondrian SP Universal Cartridge User Guide M01265 the Mondrian SP Cartridge User Guide M01344 the Mondrian SP Workstation User Manual Part No M01264 and the Mondrian SP Workstation User Manual M01322 The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to modify reverse engineer resell repackage or further sublicense under these patent applications and any patents issuing from these patent applications to use this prod uct and methods accompanying this user guide for research and development purposes solely in accordance wit
11. Tel 888 654 6544 The Netherlands U Toll Free Fax 888 296 6544 Tel 31 13 5780215 E ete imagine more from lesse custserv nugeninc com Fax 31 13 5780216 techserv nugeninc com europe nugeninc com 2012 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugeninc com NuGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners For research use only M01294 v4
12. a well lit area using a desktop lamp Alternatively the Filler Fluid containing cartridge may be inserted into the deck of the Mondrian SP or SP Workstation The reagents and samples can then be loaded while the cartridge sits in the workstation deck 1 Load 1 5 uL of each Adaptor Mix L2V9DR BC1 8 or L2V9DR BC9 16 into the appro priate port A1 through A8 matching the sample to be barcoded ensuring that the adaptors are carefully dispensed at the very bottom of the port Adaptor ports A1 through A8 are highlighted with yellow rims COOTS00 E Note If the adaptor droplet appears to be floating above the surface of the bot tom plate of the cartridge use a clean pipette tip to gently push the droplet down to the surface IV Protocol 20 Ovation SP Ultralow Library Systems Load 6 uL Ligation Master Mix into D5 Port D5 is highlighted with an D5 orange rim O Load 6 uL End Repair Master Mix into D6 Port D is highlighted with a D green rim O Load 6 uL End Repair Enhancer Master Mix into D7 Port D7 is high D7 lighted with a blue rim O Load 50 uL Bead Binding Solution in E4 Port E4 is not highlighted in E4 any way O Load 50 uL Elution Buffer into E5 Port E5 is highlighted with a grey rim E5 Load 50 uL Bead Wash Solution into E7 Port E7 is highlighted with a E7 black rim Load 50 uL of sample mix into S1 S8 ensure that the 10 minute incubation step as described above is performed prior to load
13. amples when beginning experiments and or using a new source of samples For RNA based experiments such as RNA Seq we recommend the use of the MicroArray Quality Control MAQC reference samples A and B For DNA based experiments such as WGS and Exome Sequencing we recommend the use of a control DNA sample from the HapMap project D Storage and Stability Ovation SP Ultralow Library Systems reagents are shipped in two boxes Box 1 is shipped on dry ice and should be stored at 20 C on an internal shelf of a freezer without a defrost cycle upon receipt Box 2 is shipped at room temperature but contains compo nents with multiple storage temperature requirements and should be unpacked immedi ately upon receipt e Vials labeled Agencourt RNAClean XP Beads clear cap should be removed from the top of the Box 2 shipping carton upon delivery and stored at 4 C 4 Introduction Ovation SP Ultralow Library Systems e All other Box 2 components should be stored at room temperature The kit has been tested to perform to specifications after as many as four freeze thaw cycles Kits handled and stored according to the above guidelines will perform to specifications for at least six months E Material Safety Data Sheet MSDS An MSDS for this product is available on the NuGEN website at www nugeninc com nugen index cfm support user guides ll Components A Reagents Provided Table 1 Ovation SP Ultralow DR Multiplex System 1 8
14. ary droplet Withdraw the pipette tip from the port Transfer the collected library droplet the 700 nL droplet should be visible in the pipette tip into a PCR tube or PCR plate for downstream processing Repeat steps 2 7 above if the prepared library was not captured the first time Please note that if several extraction steps are performed the additional or excess oil in the collection tube does not impact downstream processes as long as the prepared library droplet is present Continue as above with the next library until all eight libraries have been collected in separate tubes Collection of prepared libraries may also be performed by removing the cartridge from the instrument deck but it is critical to ensure that the cartridge is kept level while being moved carefully to a level surface After all of the prepared libraries have been extracted from the cartridge push the locking lever away from you to disengage the cartridge and remove the cartridge from the instrument deck by pulling it forward if not already removed Dispose of the cartridge as appropriate in laboratory waste Proceed immediately to Section V Library Enrichment via PCR V Library Enrichment via PCR 22 Ovation SP Ultralow Library Systems A Overview This section details how to prepare Ovation SP Ultralow Library Systems libraries that have been generated on the Mondrian SP Cartridge for PCR enrichment B Library Enrichment via PCR Protocol P
15. d minimize the amount of adaptor dimer in the library Does NuGEN provide reagents for performing the fragmentation step of the protocol We suggest that the end users utilize the Covaris instrument as indicated in the materials section of this user guide NUGEN does not provide the reagents used in the fragmentation steps Vill Appendix 29 Ovation SP Ultralow Library Systems Q9 Q10 Q11 Q12 Q13 Which NuGEN amplification system kits can be used to produce dsDNA for input to the Ovation SP Ultralow Library Systems The Ovation WGA FFPE System Part No 6200 Ovation RNA Seq System V2 Part No 7102 Ovation RNA Seq FFPE System Part No 7150 and Ovation 3 DGE System Part No 7200 have been specifically designed for NGS applications The cDNAs produced from these systems are compatible with the Ovation SP Ultralow Library Systems Are the Ovation SP Ultralow libraries compatible with downstream target capture methods like Agilent s SureSelect Yes the PCR enriched Ovation SP Ultralow libraries are compatible with downstream target capture methods such as Agilent s SureSelect kits Customers who wish to perform target selection on their Ovation SP Ultralow libraries should use the Encore Target Capture Module Part No 0332 in addition to the Agilent product How can gel purification be eliminated from the workflow and still pre vent adaptor dimer formation The Ovation SP Ultralow Library S
16. direct indirect consequential or incidental damages arising from the use results of use or inability to use its products NUGEN reserves the right to change alter or modify any product to enhance its performance and design NuGEN s products are developed designed and sold FOR RESEARCH USE ONLY This product is not to be used for diagnostic or therapeutic purposes nor is it to be administered to humans or animals Except as expressly set forth herein no right to modify reverse engineer distribute offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NUGEN product The buyer agrees to use NUGEN products accompanying the product insert in accordance with the intended use and the written instructions provided Table of Contents Contents Ik MNEFOMUCHON ss ccissssscsssessssssessssvecsescsssssssstecasessessesssssssvesasovsaassssuscnsasesssvecadssssseassassess 1 As Backgrounder e E E sands A E E E 1 B Performance Specifications es cssic cacsssncveusesacicessccssecchdesvncsavbesschcvavucenslechevachees 3 C OwalityCOmerol l a ere ar EEEE 3 D Storage and Stability c ciccccasicsacssiecescucesnncssevidees conse aieas 3 E Material Safety Data Sheet MSDS cecceeceeceeeeeeeeeeeeseceeeceeeeeeeeeaeeneeeeeneees 4 Ii COMPONCNES lt 5 0 scssescesscctessscessatesascucssavssvecuesdsvesassecsesvesassusaedsesvasstactstavaseseccessoaseseess 5 A Reagents Provided ceecceesceseceesneeseecenersatese
17. e template genome eukaryotic vs prokaryotic Experiments requiring more data such as WGS and from more complex genomes such as eukaryotes will require the use of more dsDNA NUGEN recommends at least 10 ng as template in the Ovation SP Ultralow assay The resulting libraries are suit able for a wide range of sequencing applications including RNA Seq Digital Gene Expression DGE genomic DNA exome sequencing amplicon sequencing ChIP Seq and more As shown in Figures 1A 1B and 1C the streamlined workflow consists of six steps 1 Fragmentation of either genomic DNA or double stranded cDNA to produce assay template 2 Addition of template and reagents to the Mondrian SP Universal Cartridge see Figure 1A 3 Hands free automation of the following assay steps on the Mondrian SP or SP Workstation see Figure 1B d Sample concentration e End repair e Sample purification e Adaptor ligation with optional sample multiplexing e Sample purification 4 PCR enrichment of the purified library see Figure 1C 5 Enriched library purification and quantification 6 Cluster formation and sequencing Introduction Figure 1 Diagram depicting the six steps of the Ovation SP Ultralow Library Sys tems workflow A Steps 1 2 Covaris fragmentation of the dsDNA template and loading the Mondrian SP Cartridge with template and reagents B Step 3 assay steps automated on the Mondrian SP Cartridge C Steps 4 6 library enrichment enriched
18. e tube Load 50 uL of Elution Buffer into port E5 of the Mondrian SP Cartridge When adding sample or reagent lower the pipette tip to the bottom of the port Do not press the tip into the bottom of the cartridge if the tip contacts the bottom of the cartridge withdraw the pipette tip slightly upwards Slowly depress the plunger to the first stopping point to dispense the reagent completely from the pipette tip do not depress the plunger completely as this will introduce bubbles into the car tridge Once all of the reagent is dispensed from the pipette tip pull the pipette tip back out of the port and dispose of the tip Note Do NOT add any samples or other reagents to the cartridge at this time Ensure that only Elution Buffer has been loaded If the cartridge is not already inserted into the Mondrian SP or SP Workstation deck carefully transport the cartridge to the Mondrian SP or SP Workstation and insert the cartridge into the deck If not already ON locate the workstation ON OFF switch at the back of the workstation and turn ON Press the On button Figure 4 on the front of the workstation to turn the workstation on Figure 4 The Mondrian SP or SP Workstation On button located on the front of the workstation 10 11 12 13 Pull the cartridge lever of the Mondrian SP or SP Workstation forward to the locked position and close the lid of the workstation Select Run on the touch screen me
19. eceaeeceeceaeeesaeesaeeesaeeeeeeeneseneeee 5 B Additional Equipment Reagents and Labware cccecesececeeeseeeeeeeneees 7 lll Planning the Experimenti lt cc scccccccsesseecicecsecseccceecsececceesepecssccsssessosscasescseusscases 9 As InputiDNA ReQuirements iicscssidiccitecendstadocdcudielh ccthtvtinecs tnd bets idsicnnecceeeed 9 B Using Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 On Illumina NGS SysteMs wicca cesses ciscteseacsamsserecensneonsescnesatessnesotawssuuesooasenseessenae 9 C Amplified Library Stonage seeroete panii aia AnaS 9 IV Protocol sccscscscscsssscssccccassssssssnessasassessssensessesssssnsassescessoscucsassvensosseassssvicassncsssvssaces 10 As ON GINIGW osen eae e e EE saute tapsdvor E RE RE 10 B Protocol NOteS i iscscdinciscni seh catessde teccasnedsadedaad ceudnestcbestionsgnavatedsedaens AENEA 10 C Agencourt RNAClean XP Purification Beads ccccecceeeeeseeeeeeteeeseeteeneees 11 De DNA Fragmentation aii iiiscisesssccasteccam a a EE EAE A tessa ARENE 12 E Cartridge Quality Control Check sssini aa A ER 12 F Protocol for the Ovation SP Ultralow Library Systems on the Mondrian SP Ca rtrid eat fat vncs zvssvascendecuncssietondssotaneus sua hasaaudd RET Aian a 15 V Library Enrichment via PCR cccccccsssscssssssssssesessseseessssssceeesseseeeseeesesseseeeseeeeas 22 A QU GIVIOW rareo entered set a E sve last canted sseneescniss Poussavcinabe EN EEE a 22 B Library Enrichm
20. end be per formed prior to adding samples and reagents to the Mondrian SP Cartridge This QC check confirms the functionality of the Mondrian SP Cartridges prior to use The Mondrian SP Cartridge QC Protocol requires inserting the Filler Fluid containing cartridge into the deck of the Mondrian SP or SP Workstation Some customers may find it easier to place the fluid containing cartridge on the workstation and then pipet the Elution Buffer into the cartridge Alternatively it may be easier to pipet the Elution Buffer into the fluid containing cartridge while the cartridge rests on the bench top and then insert the reagent and fluid filled cartridge into the deck of the Workstation moving carefully to avoid spilling the fluid or disturbing the reagent 1 Locate the Cartridge Loading Guide that is provided with each Mondrian SP Cartridge and place the loading guide on the cartridge Note it is only possible to place the loading guide on the cartridge in a single orientation 2 Optional Carefully move the Filler Fluid containing cartridge and its Cartridge Loading Guide to the deck of the Mondrian SP or SP Workstation and insert the cartridge into the deck IV Protocol 13 Ovation SP Ultralow Library Systems Remove the Elution Buffer reagent tube shipped and stored at room temperature from the Ovation SP Ultralow Library System kit Set a 50 or 100 uL single channel pipette to 50 pL and remove 50 uL of Elution Buffer from th
21. ent vi PGR Protocol s ssessisssccdesiagetiaeese sh dsanscdeastsoeduessendevenecesd 22 VI Enriched Library Purification sses sce ecctess Sessed his idaasesccbvuceetuaes Redevetzstecanspetasces 23 Pec OVENI EW odie sh25G oxi A N E R cued decereaccesaade vay sanees A ARRS 23 B Enriched Library Purification Protocol isena ae 23 C Quantitative and Qualitative Assessment of the Purified Enriched Libraries 24 VIL Technical SUPPOFT ores erresis sensein a ea 26 VIILA PD ONGIK sve cccscstesecicdevisnccsiecevsvenscusdenieesccusseusvenacusdevvonbacvescvetnnscvedeviangiesesevvdeausueies 27 A Sequences of the DR Barcodes in the Multiplexed Reactions 27 B Frequently Asked Questions FAQS cece tees eens te cee teeeteeeseeesneeeseeees 28 C Update HiO esperinta r atest ES 30 1 Introduction Ovation SP Ultralow Library Systems A Background The Ovation SP Ultralow Library Systems composed of the Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 are a complete reagent cartridge and protocol package for the simple automation on the Mondrian SP or SP Workstations of DNA library preparation protocols used in next generation sequencing These systems enable library preparation starting with as little as 1 0 ng of sheared double stranded DNA dsDNA The recommended amount of dsDNA template for the Ovation SP Ultralow assay depends on several factors including the experiment itself RNA Seq vs WGS and the complexity of th
22. f 1 2 3 4 Binding of DNA to Agencourt RNAClean XP beads Magnetic separation of beads from supernatant Ethanol wash of bound beads to remove contaminants Elution of bound DNA from beads Figure 3 Agencourt RNAClean XP Bead purification process overview 1 Binding 2 Separation 3 Ethanol Wash 4 Elution Magnet Magnet Magnet Reproduced from original picture from Agencourt Beckman Coulter Genomics Additional Tips and Notes Remove beads from 4 C and leave at room temperature for at least 15 minutes before use and ensure that they have completely reached room temperature Cold beads reduce recovery Fully resuspend the beads by inverting and tapping before adding to sample Note that the ratio of Agencourt RNAClean XP bead volume to sample vol ume for the bead purification of the amplified library differs from the standard Agencourt protocol It is critical to let the beads separate on the magnet for a full 5 minutes Removing binding buffer before the beads have completely separated will impact DNA yields After completing the binding step it is important to minimize bead loss when removing the binding buffer With the samples placed on the magnet remove only 90 uL of the binding buffer from each sample for bead purification of the IV Protocol 12 Ovation SP Ultralow Library Systems amplified material Some liquid will remain at the bottom of the tube but this will minimize bead loss e Any significant
23. ge on a level surface and fill the cartridge with filler fluid according to the instructions in the Mondrian SP Universal Cartridge User Guide M01265 P01198 v1 IV Protocol 19 Ovation SP Ultralow Library Systems Next follow the instructions below to load reagents into their appropriate cartridge ports Use a 10 uL pipette to add all reagent master mixes and the adaptors It is criti cal that a 10 uL pipette be used to add the adaptors to the cartridge as a 2 uL pipette will not generate sufficient force to expel the adaptors from the pipette tip into the filler fluid containing cartridge Use a 100 or 200 uL pipette for adding the samples Bead Binding Solution Elution Buffer and Bead Wash Solution Load the ports in a steady manner to avoid overflow of the filler fluid When adding sample or reagent lower the pipette tip to the bottom of the port Do not press the tip into the bottom of the cartridge if the tip contacts the bottom of the cartridge withdraw the pipette tip slightly upwards Slowly depress the plunger to the first stopping point to dispense the reagent completely from the pipette tip do not depress the plunger completely to the second stopping point as this will introduce bubbles into the cartridge Once all of the reagent is dispensed from the pipette tip pull the pipette tip back out of the port and dispose of the tip Note that sample and reagent loading may be more easily performed in
24. h the intended use described and the written instructions provided in this user guide No license to make or sell products by use of this product is granted to the buyer whether expressly by implication by estoppels or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research please contact NUGEN Technologies Inc 201 Industrial Road Suite 310 San Carlos CA 94070 Phone 888 654 6544 or 650 590 3600 FAX 888 296 6544 or 650 590 3630 Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase provided that the product is handled and stored according to published instructions and that the product is not altered or misused If the product fails to meet these performance standards NuGEN will replace the product free of charge or issue a credit for the purchase price NUGEN s liability under this warranty shall not exceed the purchase price of the product NUGEN shall assume no liability for
25. ing onto the cartridge Sample input ports are highlighted with red OOOOS00O0 If the cartridge is not already on the Mondrian SP or SP Workstation deck care fully transport the cartridge to the Mondrian SP or SP Workstation insert the cartridge into the deck pull the locking lever forward and close the lid PO1198 v1 Mondrian SP or SP Workstation Initialization Instructions is If not already ON locate the workstation ON OFF switch at the back of the work station and turn ON Press On button on the front of the workstation Select Run from the touch screen menu select the Ovation SP Ultralow protocol and follow the instructions on the screen to begin the run IV Protocol 21 Ovation SP Ultralow Library Systems Library Collection from the Mondrian SP Cartridge 1 2 3 10 11 Open the Mondrian SP or SP Workstation lid if closed after completion of the run Set a 100 or 200 uL pipette to collect 20 uL Depress the pipette plunger before placing the pipette tip in the first collection port of the ports marked Sample Collection o SPereseeerers Insert the pipette tip into the collection port so that the tip is touching the bottom surface of the port sealing the pipette tip Release the plunger and then lift the pipette tip up 1 mm from the bottom sur face ensure the tip remains in the collection port Fluid will quickly fill the pipette tip including the libr
26. l amounts of reagents into the reaction mix pipet up and down several times to ensure complete transfer e When instructed to pipet mix gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix e Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block e Components and reagents from other NUGEN kits should not be used with the Ovation SP Ultralow Library Systems e Use only fresh ethanol stocks to make 70 ethanol used in the final purification protocol e Make the ethanol mixes fresh as well carefully measuring both the ethanol and water Lower concentrations of ethanol in the wash solution will result in loss of yield as the higher aqueous content will dissolve the DNA and wash it off the beads IV Protocol 11 Ovation SP Ultralow Library Systems C Agencourt RNAClean XP Purification Beads Tips and Notes Relevant to the Enriched Library Purification Protocol Section VI Part B There are significant modifications to the Agencourt RNAClean XP beads standard pro cedure therefore you must follow the protocols outlined in this user guide for the use of these beads However you may review the Beckman Coulter Genomics user guide to become familiar with the manufacturer s recommendations for handling the beads The bead purification process used for cDNA purification before amplification consists o
27. l times Mondrian SP A prob Press OK on the Run Complete screen to return to cartridge failed lem was the main menu Remove the cartridge from the deck Remove cartridge detected and set it aside do not discard and contact NUuGEN from instru with Technical Support for further instructions Users who ment deck and droplet proceed with SP protocols and or who load additional set aside prior transport SP reagents and samples onto failed cartridges are to contacting within the doing so at their own risk and will not be compensated NuGEN Technical cartridge for loss of reagents samples or cartridges by NuGEN Support Technologies Inc ondrian SP The results Repeat the Mondrian SP Cartridge QC protocol one cartridge status are incon more time is undetermined clusive and Pleaseconsult must b e Do not add any additional Filler Fluid or Elution Buffer ondrian repeated to the cartridge SP Universal one more e Press OK on the Run Complete screen to return to the Cartridge User time prior main menu Shee elas to making e Proceed to re run the Mondrian SP Cartridge QC pro prats MUGEN 3 determi tocol starting from Step 10 in the protocol above SP Library nation on Systems User the quality If the message after the second Mondrian SP Cartridge Guide for further of the QC protocol is instructions cartridge e Mondrian SP cartridge passed then proceed as out lined in Next Step for passing cartridges
28. library purification plus library quantification then cluster formation and sequencing Mondrian SP Cartridge ati Step 2 Filler E4 ES E7 Make master mix Fluid oe 0 o load in reagent ports 0 88 Step 1 Sample Collection 21 0 ng fragmented DNA generated ona Covaris instrument Single use only Sample Input P E Ovation SP Ultralow Library System reagents D CR R R RRE E o2 a 4 5 6 7 8 Adaptors eee e868 1 2 3 4 5 6 7 Ovation SP Ultralow Library System Step 3 Mondrian SP or SP Workstation performs the following steps in 3 hours End repair steps 1 and 2 Add adaptors and ligate optional multiplexing z Workstation Steps 4 6 Step 4 Performed PCR enrichment IUT off Mondrian of purified library SP or SP Sample purification Step 5 Library quantification Library quantification ae 6 AATCGGATCGGTAGGAT Cluster formation scrcGATGCAAGTGATC and sequencing GTAGCAAAATCCTGAGA 2 Ovation SP Ultralow Library Systems Introduction This product contains components with multiple 3 storage temperatures Ovation SP Ultralow Library Systems The entire workflow requires only one manual bead purification step and no gel purification Starting with as little as 1 ng of fragmented dsDNA the protocol can be completed in approximately 4 5 hours and produces libraries ready for cluster forma tion and eithe
29. lications Can the Ovation SP Ultralow Library Systems be used with paired end sequencing Yes they can be used for both single end and paired end sequencing Special consideration should be given to the expected insert size in the paired end assay Is there a lower or an upper size limit that can use to make my library NuGEN Technologies Inc has successfully used fragments ranging from 200 to 500 bp in size It is possible to generate a library using both smaller and larger fragments although a specific upper and lower size limit has not been established How much material should load into the cBot Please follow manufacturer s recommendations for library QC quantitation balancing and loading of the amplified library on the cBot Do the Ovation SP Ultralow Library Systems work with the Illumina Cluster Station predecessor of the cBot instrument Yes the Systems are also compatible with the Illumina Cluster Station don t have access to a Covaris instrument can use alternative fragmen tation methods We have evaluated only Covaris fragmented DNA during the development of the Ovation SP Ultralow Library Systems Other mechanical means of frag mentation such as sonication may be suitable as well How does your protocol improve the efficiency of ligation and avoid adaptor dimer formation The Ovation SP Ultralow Library Systems utilize optimized chemistries to increase the efficiency of blunt end adaptor ligation an
30. lt in low amplification yield Use of DNA samples of excessively low quality or samples that are incorrectly quantitated may yield poor results B Using Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 on Illumina NGS Systems The Ovation SP Ultralow Multiplex Systems 1 8 and 9 16 use a Dedicated Read DR aka second sequencing primer approach for multiplex sequencing Figure 2 depicts the DR multiplex barcode strategy Figure 2 Dedicated read multiplexing strategy used by the Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 Dedicated Read Barcode Design Illumina Standard Seq Primer Library Insert Illumina Index Seq Primer Flow cell surface m The Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 use the same approach to multiplexing utilized in the standard Illumina method These libraries should be sequenced using the Illumina protocol for multiplex sequencing The DR barcode sequences are found in Appendix A of this user guide and must be entered into the Illumina software prior to the analysis C Amplified Library Storage Amplified libraries may be stored at 20 C IV Protocol 10 Ovation SP Ultralow Library Systems A Overview The library preparation process used in the Ovation SP Ultralow Library Systems is per formed in two stages The first stage sample concentration DNA end repair purifica tion adaptor ligation and purification occurs on the Mondrian SP or SP Worksta
31. nu choose the Mondrian SP Cartridge QC protocol from the list of protocols and then select Next to proceed to the Protocol Information screen Select Next to proceed to the Run Information screen Optional Enter run details as required in the Run Information screen Select Next to proceed to the Run Confirmation screen and select Start Run The Mondrian SP Cartridge QC protocol will take about nine minutes to complete During this test Elution Buffer droplets will be dispensed from the E5 port and trans IV Protocol 14 Ovation SP Ultralow Library Systems ported around the cartridge prior to being discarded The purpose of this test is to confirm the successful transport of droplets across all lanes of the cartridge At the end of the protocol the instrument will display the Run Complete screen and one of the following messages MESSAGE MEANING NEXT STEP Mondrian No errors Press OK on the Run Complete screen to return to the SP cartridge detected main menu Proceed to section C Loading Samples and passed Continue Droplet Reagents below in the Mondrian SP Universal Cartridge to intended transport user guide or follow the instructions for 3rd party protocol was normal reagents in the appropriate applications note The user may remove the cartridge from the Workstation to load samples and remaining reagents on the bench top taking care that the cartridge remains level at al
32. or 0 2 mL PCR tube accord ing to the recipe in Table 4 End Repair Master Mix below 3 Mix well by carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube IV Protocol 16 Ovation SP Ultralow Library Systems Table 4 End Repair Master Mix COMPONENT VIAL CAP VOLUME End Repair Buffer Mix ER1 ver 5 Blue 9 0 uL End Repair Enzyme Mix ER2 ver 4 Blue 1 0 uL Total volume 10 0 pL Prepare End Repair Enhancer Master Mix 1 Thaw End Repair Enhancer Buffer Mix ER4 at room temperature vortex to mix well and spin down briefly Keep End Repair Enhancer ER3 on ice 2 Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube accord ing to the recipe in Table 5 End Repair Enhancer Master Mix below 3 Mix well by carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube Table 5 End Repair Enhancer Master Mix COMPONENT VIAL CAP VOLUME End Repair Enhancer Buffer Mix ER4 Blue 7 0 uL End Repair Enhancer ER3 Blue 3 0 uL Total volume 10 0 pL Prepare Ligation Master Mix ily Thaw Ligation Buffer Mix L1 ver 5 at room temperature and vortex to mix well Keep Ligation Enzyme Mix L3 ver 4 on ice Prepare master mix in a 0 5 mL microcentrifuge tube or 0 2 mL PCR tube accord ing to the recipe in Table 6 Ligation Maste
33. ow to use Agencourt RNAClean XP beads to purify the enriched libraries prepared in the preceding section B Enriched Library Purification Protocol Note For this step we recommend using either an Agencourt SPRIPlate Ring Magnet or the Agencourt SPRIStand iP 10 11 Remove the aqueous phase approximately 50 uL liquid layer in the bottom of the tube from each of the PCR reaction vessels and transfer to a clean tube or PCR plate Ensure the Agencourt RNAClean XP beads have completely reached room tem perature before proceeding Resuspend beads by inverting and tapping the tube Ensure beads are fully resus pended before adding to sample After resuspending do not spin the beads An excess of beads is provided therefore it is not necessary to recover any trapped in the cap Prepare a 70 ethanol wash solution At room temperature add 50 uL 1 volume of the bead suspension to each reac tion Mix by pipetting up and down 10 times It may be helpful to use a multi channel pipettor to ensure the incubation times are uniform Incubate at room temperature for 10 minutes Transfer tubes to magnet and let stand 5 minutes to completely clear the solution of beads Carefully remove only 90 uL of the binding buffer and discard it Leaving some of the volume behind minimizes bead loss at this step Note The beads should not disperse instead they will stay on the walls of the tubes Significant loss of beads at this stage
34. r Mix below Mix well by carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube 17 IV Protocol Table 6 Ligation Master Mix COMPONENT VIAL CAP VOLUME Ligation Buffer Mix L1 ver 5 Yellow 7 0 uL Ligation Enzyme Mix L3 ver 4 Yellow 3 0 uL Total volume 10 0 pL Prepare Adaptors 1 Thaw ligation adaptors at room temperature vortex to mix well and spin down briefly The adaptors will be loaded onto the cartridge as indicated below Step 1 of the Mondrian SP Cartridge Loading Instructions Ovation SP Ultralow Library Systems IV Protocol 18 Ovation SP Ultralow Library Systems 3 Mondrian SP Cartridge Loading Instructions Figure 5 Mondrian SP Cartridge loading guide for the Ovation SP Ultralow Library System protocol Va EO OQO e OO OOOOC0O0OO Serer seer ars COODS00 G Ovation SP Ultralow Library System Single use only 60009000 O 1 2 3 4 5 6 7 8 Loading Reagents and Samples in the Mondrian SP Cartridge If the Mondrian SP Cartridge integrity has been confirmed through the Cartridge QC Protocol then it is not necessary to add any additional Filler Fluid to the cartridge prior to loading samples and reagents If the Cartridge QC Protocol has not been per formed then it is necessary to add Filler Fluid to the cartridge prior to loading samples and reagents Place the cartrid
35. r single read or paired end sequencing In addition to genomic and other double stranded DNA sources the Ovation SP Ultralow Library Systems have been designed for seamless integration with NUGEN s Ovation WGA FFPE System Part No 6200 Ovation RNA Seq V2 RNA Seq FFPE Encore Target Capture Module and Prokaryotic RNA Seq Systems Part Nos 7102 7150 0332 and 9030 respectively to enable a complete end to end solution for transcriptome library construction starting with total RNA The Ovation SP Ultralow DR Multiplex Systems 1 8 Part No 8033 and 9 16 Part No 8034 each provide eight unique dedicated read barcoded adaptors to prepare libraries for multiplex sequencing Together these two multiplex kits enable up to 16 plex sequencing B Performance Specifications The Ovation SP Ultralow Library Systems are designed to produce DNA libraries suit able for either single read or paired end sequencing on the Illumina Genome Analyzer Ikx Ile GAII MiSeq HiScan SQ or HiSeq NGS platforms without gel based size selection using 1 100 ng input of double stranded DNA The Ovation SP Ultralow Library Systems generate libraries suitable for loading onto an Illumina cBot Cluster Generation System in about 4 5 hours C Quality Control Every lot of the Ovation SP Ultralow Library Systems undergoes functional testing to meet specifications for library generation performance NuGEN Technologies Inc recommends the use of control s
36. repare Library Enrichment Master Mix 1 Thaw Amplification Buffer Mix P1 ver 2 Amplification Primer Mix P2 ver 5 DMSO P4 and Nuclease free Water D1 at room temperature and vortex to mix well Keep Amplification Enzyme Mix P3 on ice Prepare Library Enrichment Master Mix in a 1 5 mL Eppendorf tube according to the recipe in Table 7 Mix well by carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube Keep the prepared Library Enrichment Master Mix on ice Table 7 Library Enrichment Master Mix for 8 Libraries COMPONENT VOLUME Amplification Buffer Mix P1 ver 2 193 0 uL Amplification Primer Mix P2 ver 5 22 0 uL Amplification Enzyme Mix P3 6 0 uL DMSO P4 22 0 uL Nuclease free Water D1 198 0 uL Total volume 441 0 pL and mix well ing program Add 50 uL Library Enrichment Master Mix to each collected library sample tube Seal and place tubes or plate in a thermal cycler programmed to run the follow 72 C 2 min 15 cycles 94 C 30 sec 60 C 30 sec 72 C 1 min 72 C 5 min hold at 10 C Cycle number may be optimized depending on the input DNA quantity For example 1 ng DNA inputs may require up to 18 cycles 50 ng DNA inputs may require only 10 12 cycles VI Enriched Library Purification 23 Ovation SP Ultralow Library Systems A Overview This section details h
37. tion and takes approximately 3 hours to complete The second stage library enrichment PCR and enriched library purification occurs separately from the Mondrian SP or SP Workstation and takes approximately 1 5 hours to complete The total time to pre pare amplified and purified libraries that are ready for sequencing is approximately 4 5 hours Protocol Notes e The system is designed and intended for processing eight samples at a time Do not attempt to prepare smaller volume master mixes or process fewer than eight samples using the Ovation SP Ultralow Library System e We recommend the routine use of a positive control DNA Especially the first time you set up a reaction the use of a positive control DNA will allow the establishment of a baseline of performance and provide the opportunity to become familiar with the bead purification steps e Use the water provided with the kit green cap vial D1 or an alternate source of nuclease free water We do not recommend the use of DEPC treated water with this protocol e Thaw components used in each step and immediately place them on ice Do not thaw all reagents at once e Always keep thawed reagents on ice unless otherwise instructed e After thawing and mixing buffer mixes if any precipitate is observed re dissolve it completely prior to use You may gently warm the buffer mix for 2 minutes at room temperature followed by brief vortexing Do not warm any enzyme mixes e When placing smal
38. ystems workflow uses bead based purifi cation and efficient primer design thus eliminating the need for gel based purification Can I reduce the number of PCR amplification cycles recommended by the Ovation SP Ultralow Library Systems workflow by using more than 1 ng fragmented DNA for input Yes it is possible to reduce the number of PCR amplification cycles in the Library Enrichment PCR when using more than 1 ng of input DNA as starting material Can I use the Ovation SP Ultralow Library System with the Mondrian SP Workstation Yes the Ovation SP Ultralow Library System kit can be used on both the Mondrian SP and SP Workstation Make sure to select the correct protocol Ovation SP Ultralow protocol from the Workstation menu when running this kit on the SP Workstation VIII Appendix i C Update History This document the Ovation SP Ultralow Library System User Guide M01294 v4 includes the following updates DESCRIPTION SECTION PAGE S Removed references to Ovation SP Ultralow Library Th hout Th hout System Part No 8030 he ata eee Corrected part number and location for Agencourt ILA 57 RNAClean XP Beads ai Updated reagent volumes for Sample Solution IVE 16 Preparation B NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 109 For our international distributors contact San Carlos CA 94070 USA 9350 AC Leek information visit our website Toll Free
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