User Guide
Contents
1. 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 l Introduction nesenas e E netneseeacesessosiacassotessasonsas 1 As Background esisin aine a nae E E E EE E ERNE 1 B Performance Speciticationss x lt ssessseisisthsabisscesszecssescalecesyeabeaes E 3 C SOuality Control sssini sanai E EEEE E aa 3 Dy Storagsand Stabilityssser direi e aA ARE EESE 4 E Material Safety Data Sheet MSDS sssssssissssississssisrisisrissistsrisssisrisristsrrsrese 4 ll CONMMPOMGMES 55s cnda5e5865 lt cxdasseces occesesescs E EEr EE RESE EAEE ENE EE EKENS 5 Ay Reagents Provided asenisman suaneateceesaceseacusdneniaiesne N aee E EEEN a Ean 5 B Additional Equipment Reagents and Labware sssssssseseisisisersisisnesrnsisinei 7 Ill Planning the Experiment ccccccccsssssssseesesssessesseese2. Figure 5 shows a cutaway side view sche matic of a cartridge VII Appendix 26 Ovation SP Ultralow Library Systems Figure 5 Cartridge cutaway side view Cartridges Top plate lt Droplet Filler fluid oil SS or Insulating polymer PCB substrate Electrode Hydrophobic coating Cartridges are one time use only Cartridge re use is not supported even if a run is canceled prior to completion Figure 6 Top view of a Mondrian SP Cartridge Sample ports along the bottom edge are labeled S1 8 Other reagent port rows are labeled A E Note that there is no row B There are eight ports in rows A and C and seven in rows D and E The port labeled F is sized for a standard Luer lock connector through which the Filler Fluid is added to the cartridge The cartridge electrodes are visible as rows of small gold squares at the top of the cartridge F Filler Fluid port E Reagents and master mixes D Reagents and master mixes C Collection of final purified libraries A Adaptor ports Bubble trap ports nothing is added or removed via these ports Sample input ports VII Appendix The SP Filler Fluid has been degassed and pack 27 aged in a sealed single use vial Do not open the vial until ready for use Ovation SP Ultralow Library Systems Filling the Cartridge SP Cartridges must be filled with the provided Filler Fluid prior to loading with reagents This process should be
3. carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube 4 Keep the prepared Library Amplification Master Mix on ice 16 Ovation SP Ultralow Library Systems IV Protocol 17 Ovation SP Ultralow Library Systems Table 6 Library Amplification Master Mix label tube E2 COMPONENT VIAL CAP VOLUME Amplification Buffer Mix P1 ver 5 Red 14 2 uL Amplification Primer Mix P2 ver 11 Red 6 7 uL DMSO P4 Brown 1 6 uL Amplification Enzyme Mix P3 ver 4 Red 2 5 uk Total volume 25 0 pL 3 Mondrian SP Cartridge Loading Instructions Loading Reagents and Samples in the Mondrian SP Cartridge If you have confirmed the integrity of the Mondrian SP Cartridge through the Mondrian SP Cartridge OC Protocol it is not necessary to add any additional Filler Fluid to the cartridge prior to loading samples and reagents If the Mondrian SP Cartridge QC Protocol has not been performed you must add Filler Fluid to the cartridge prior to loading samples and reagents Place the cartridge on a level surface and fill the cartridge with filler fluid according to the Mondrian SP Cartridge Handling instructions in Appendix B IV Protocol 18 Ovation SP Ultralow Library Systems Figure 3 Cartridge loading guide for the Ovation SP Ultralow Library System protocol E2 E3 E4 OOOOC 500 D D7 0009565 SOOO Collection lere
4. or samples that are incorrectly quanti tated may yield poor results B Using Ovation SP Ultralow Library Systems on Illumina NGS Systems The Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 use a Dedicated Read DR or 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 Library Systems Dedicated Read Barcode Design Illumina Standard Seq Primer Library Insert Illumina Index Seq Primer sacode m Flow cell surface The Ovation SP Ultralow Library Systems use the same approach to multiplexing used in the standard Illumina method These libraries should be sequenced using the Illumina protocol for multiplex sequencing The DR barcode sequences are found in 8 Ovation SP Ultralow Library Systems Ill Planning the Experiment 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 9 Ovation SP Ultralow Library Systems IV Protocol 10 Ovation SP Ultralow Library Systems A Overview The library preparation process used in the Ovation SP Ultralow Library Systems is performed on the Mondrian SP Workstation and takes 6 7 hours to complete After collection of the droplets from the Mondrian SP Cartridge we recommend perform ing librar
5. owners For research use only M01327 v4
6. source of samples For RNA based experiments such as RNA Seq we recom mend using the MicroArray Quality Control MAQC reference samples A and B For DNA based experiments such as WGS and exome sequencing we recommend using a commercial source of genomic DNA Introduction This product contains components with multiple 4 storage temperatures Ovation SP Ultralow Library Systems 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 Box 2 is shipped at room temperature but contains components with multiple storage temperature requirements and should be unpacked immediately 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 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 5 Components Ovation SP Ultralow Library Systems A Reagents Provided Table 1 Ovation SP Ultr
7. 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 direct indirect consequential or incidental damages arising from the use
8. 61876 676 5555568 Adaptors Ovation SP Ultralow DR Multiplex Systems Single use only ga NUGEN imagine mare fram less ee ee Input OOCO90CO 8 Follow the instructions below to load reagents into their appropriate cartridge ports e Use a 10 or 20 uL pipette to add the adaptors and all reagent master mixes except for the Library Amplification Master Mix Use a 100 or 200 uL pipette for adding the samples Bead Binding Solution Elution Buffer Library Amplification Master Mix 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 com pletely from the pipette tip As you raise the pipette tip from the port you should complete a gentle blow out by fully depressing the pipette plunger just prior to exiting the filler fluid to ensure all the reagent is dispensed Note Using a desktop lamp to illuminate your work area may facilitate sample and reagent loading We recommend loading the reagents and samples with the cartridge on the deck of the Mondrian SP Workstation IV Protocol 1 Load 25 uL Library Amplification Master Mix into port E2 white rim
9. E2 2 Load 50 uL Bead Binding Solution into ports E3 and E4 no color E3 E4 3 If cartridge QC has not been completed load 50 pL Elution Buffer into E5 port E5 grey rim If cartridge QC has been completed skip this step and proceed to step 4 4 Load 50 uL Bead Wash Solution into ports E6 and E7 black rim E6 E7 OO 5 Load 8 uL Ligation Master Mix into port D5 orange rim D5 O 6 Load 8 uL End Repair Master Mix into port D green rim D O 7 Load 8 uL End Repair Enhancer Master Mix into port D7 blue rim D7 O 8 Load 1 5 uL of each DR Multiplex Ligation Adaptor Mix L2V13DR BC1 8 or L2V13DR BC9 16 into the appropriate port A1 through A8 yellow rims matching the sample to be barcoded ensuring that the adaptors are carefully dispensed at the bottom of the port WV O000000 Adaptors 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 19 Ovation SP Ultralow Library Systems IV Protocol 20 Ovation SP Ultralow Library Systems 10 Load 50 uL of sample mix into S1 S8 red rims Ensure that the 10 minute incuba tion step described above is performed prior to loading onto the cartridge Mix the samples once more before loading Sample Input 00086000 If the cartridge is not already on the Mondrian SP Workstation deck carefully transpor
10. Ovation SP Ultralow Library Systems PART NOS 8133 8134 E imagine more GEI less Patents Licensing and Trademarks 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 Workstation User Manual 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 with the intended use described and the written instructions provided in this
11. ables PCR clean Safe Lock Tube 0 5 mL U S A Cat 022431005 international Cat 0030 108 035 Disposable gloves Lint free wipes such as Kimwipes or Berkshire Super PolX 1200 Wipers VWR Cat 21914 Canned air Ice bucket Cleaning solutions such as DNA OFF MP Biomedicals Cat QD0500 To Order Affymetrix www affymetrix com Agilent www agilent com Covaris www covarisinc com Eppendorf www eppendorf com Life Technologies www lifetechnologies com MP Biomedicals www mpbio com Sorenson BioScience www sorbio com VWR www vwr com Ill Planning the Experiment A Input DNA Requirements The Ovation SP Ultralow Library Systems are designed to work with 10 to 100 ng of fragmented genomic dsDNA or ds cDNA The recommended amount of dsDNA template for the Ovation SP Ultralow assay depends the type of experiment and the complexity of the template genome Experiments involving more complex genomes require more dsDNA as template We recommend using at least 10 ng of input dsDNA for whole genome sequencing WGS of complex eukaryotic genomes DNA samples must be free of contaminating proteins RNA organic solvents includ ing phenol and ethanol and salts We recommend using a commercially available system for DNA cDNA isolation The A260 A280 ratio for DNA samples should be in excess of 1 8 Use of DNA samples with lower ratios may result in low amplification yield DNA samples of excessively low quality
12. alow DR Multiplex Systems 1 8 and 9 16 Components and Reagents Part Nos 8133 annd 8134 PART VIAL NUMBER DESCRIPTION BOX CAP VIAL 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 S01662 Ligation Buffer Mix 1 of 2 Yellow L1 ver 5 8133 DR Multiplex Ligation Adaptor 1 of 2 Yellow 8133 S01787 Mixes L2V13DR BC1 S01788 L2V13DR BC2 S01789 L2V13DR BC3 S 01790 L2V13DR BC4 S01791 L2V13DR BC5 01792 L2V13DR BC6 501793 L2V13DR BC7 S01794 L2V13DR BC8 8134 8134 S01795 L2V13DR BC9 S01796 L2V13DR BC10 501797 L2V13DR BC11 S01798 L2V13DR BC12 S01799 L2V13DR BC13 S01800 L2V13DR BC14 S01801 L2V13DR BC15 01802 L2V13DR BC16 S01467 Ligation Enzyme Mix 1 of 2 Yellow L3 ver 4 S01804 Amplification Buffer Mix 1 of 2 Red P1 ver 5 S01803 Amplification Primer Mix 1 of 2 Red P2 ver 11 S01805 Amplification Enzyme Mix 1 of 2 Red P3 ver 4 S01668 DMSO 1 of 2 Brown P4 S 01001 Nuclease free Water 1 of 2 Green D1 6 Components Ovation SP Ultralow Library Systems Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 Components and Reagents Part Nos 8133 annd 8134 continued FURIE DESCRIPTION BOX ae VIAL NUMBER P01208 Mondrian SP Cartridges x4 2of2 N A N A S01719 SP Cartridge Fil
13. and then select Start Run The Mondrian SP Cartridge QC protocol will take about 15 minutes to complete During this test Elution Buffer droplets will be dispensed from the E5 port and trans ported around the cartridge prior to being returned to port E5 The purpose of this test is to confirm the basic performance 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 SP No errors were Press OK on the Run Complete screen Cartridge passed Continue to intended protocol detected Droplet transport was normal Cartridge is ready to runa protocol to return to the main menu Proceed to the intended protocol Mondrian SP Cartridge failed Remove cartridge from instrument deck and set aside prior to contacting NuGEN Technical Support A problem was detected with droplet trans port within the cartridge Press OK on the Run Complete screen to return to the main menu Carefully remove the cartridge from the deck of the workstation setting aside the cartridge for possible return to NuGEN Begin with a new cartridge and contact NuGEN Technical Support to request a replacement for the failed cartridge F Protocol for the Ovation SP Ultralow Library Systems on the Mondrian SP Cartridge 1 Sample Solution Preparation Preparation of the sample solution
14. bit Fluorometer For information on PCR enrichment cycles please see the Mondrian SP Workstation Initialization Instructions in the protocol section of the user guide VII Appendix ney NuGEN imagine more from less F Update History This document the Ovation SP Ultralow Library System User Guide M01327 v4 has been updated from the previous version to address the following topics Description Section Page s Updated protocol for Cartridge Quality Control Check IV E 12 13 Updated volume of reagents to load in wells D5 D7 IV F 19 NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 109 San Carlos CA 94070 USA 9350 AC Leek Toll Free Tel 888 654 6544 The Netherlands Toll Free Fax 888 296 6544 Tel 31 13 5780215 custserv nugeninc com Fax 31 13 5780216 techserv nugeninc com europe nugeninc com www nugeninc com For our international distributors contact information visit our website 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
15. carried out on the bench top 1 Use scissors to cut open the Mondrian SP Cartridge pouch Remove the Mondrian SP Cartridge Note Do not attempt to tear open the pouch as this may damage the SP Cartridge Lay the cartridge on a level bench top and place the appropriate SP Library Systems Cartridge Loading Guide on the cartridge Remove the empty Filler Fluid tube and the SP Filler Fluid bottle from the NuGEN SP Library System kit Carefully grasp the tab on the red aluminium seal of the SP Filler Fluid bottle and pull up and back to remove the seal Remove the top and bottom caps from the empty Filler Fluid tube Attach the empty Filler Fluid tube to the SP Cartridge by inserting the tip of the Filler Fluid tube into the port marked Filler Fluid on the cartridge while gently twisting the tube in a clockwise direction to secure the Filler Fluid tube on the cartridge Remove the rubber stopper from the bottle of SP Filler Fluid Slowly pour the SP Filler Fluid into the Filler Fluid tube Be careful not to pour too quickly as this may introduce bubbles into the fluid Air bubbles entering the cartridge may interfere with droplet movement During the cartridge filling pro cess you will begin to see the Filler Fluid rising in the sample and reagent ports You should see Filler Fluid in all sample and reagent ports by the end of the filling process Once the entire volume of Filler Fluid has been delivered carefully
16. dict asvttesxn lt ccaheeattes O 30 E Frequently Asked Questions FAQS 0 cc cect seer cee teee tees sneseeneeeeete 31 Be Update Histo ny ssisicsssecacesassstavescusatdestensvacay ctacesasavease eaens sveeaseesseresreeeeeniaatcs 33 1 Introduction Ovation SP Ultralow Library Systems A Background The Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 Part Nos 8133 and 8134 are complete reagent cartridge and protocol packages for the simple automa tion of DNA library preparation protocols on the Mondrian SP Workstation These systems enable library preparation for next generation sequencing starting with as little as 10 ng of sheared double stranded DNA dsDNA The resulting libraries are suitable for a wide range of sequencing applications including RNA Seq genomic sequenc ing amplicon sequencing ChIP Seq and more As shown in Figure 1 the streamlined workflow consists of five steps 1 Fragmentation of either genomic DNA or double stranded cDNA to produce the assay template 2 Addition of template and reagents to the Mondrian SP Cartridge 3 Hands free automation of the following assay steps on the Mondrian SP Workstation Sample concentration e End repair e Sample purification e Adaptor ligation e Library amplification e Amplified library purification 4 Quantitation of the purified amplified library 5 Cluster formation and sequencing 2 Introduction Figure 1 The Ovation SP U
17. e control electronics and lock the cartridge into place Note Once a run has started do not move the locking lever as this will result in an unrecoverable interruption of the running protocol The Mondrian SP Workstation will not run with the lever in the up position Cleaning the Workstation Contact Pins The interface pins at the workstation cartridge interface may become dirty and cause performance issues If this happens you will need to clean the pins Materials e Lint free wipes such as Kimwipes or Super PolX 1200 Wipers Do not use cotton or any material that may leave particles behind e Isopropyl alcohol e Canned air Procedure il 2 3 Turn the workstation off and unplug from the power source Soak the wipe in isopropyl alcohol Gently insert a folded wipe into the interface pin area at the back of the cartridge deck and rub all pins with the wipe Wait 2 minutes for the isopropyl alcohol to evaporate VII Appendix 30 Ovation SP Ultralow Library Systems 5 Blow the area dry with canned air 6 Plug the workstation back in and turn it on Note We recommend cleaning the pins once a week after any prolonged storage of the workstation after a Filler Fluid spill or if the Mondrian SP Cartridge QC proto col returns a Mondrian SP Cartridge Failed or a Mondrian SP Cartridge Status Undetermined message D PCR Amplification Artifacts In some instances PCR amplification may create art
18. ed read DR adaptors used in Ovation SP Ultralow DR Multiplex System 9 16 Part No 8134 LIGATION 6 NT BARCODE BARCODE BARCODE ADAPTOR MIX SEQUENCE AS READ BY PAIRING PAIRING THE SEQUENCER 2 PLEX gt 2 PLEX L2V13DR BC9 AAGAGG Duplex Set 1 L2V13DR BC10 GGAGAA One of the duplex L2V13DR BC11 AGCATG sets from the 7 F Duplex Set 2 column to the left L2V13DR BC12 GAGTCA must beusedin L2V13DR BC13 CGTAGA combination with Duplex Set 3 any of the other L2V13DR BC14 TCAGAG remaining six indi vidual barcodes L2V13DR BC15 CACAGT Duplex Set 4 L2V13DR BC16 TTGGCA B Mondrian SP Cartridge Handling Background The Mondrian SP Cartridge is used with the Mondrian SP System and offers a convenient 8 sample batch size simple reagent loading and easy sample recovery Reagents are contained in discrete droplets encased within filler fluid isolating the reactions from the lab environment and carryover contamination Construction When prepared for use cartridges consist of an oil layer Filler Fluid sandwiched between a PCB substrate and a clear top plate The PCB substrate is patterned with insulated electrodes By changing the relative voltages of the patterned electrodes aqueous droplets are manipulated to perform complex assays Droplets are dispensed from loading ports transported to various locations on the cartridge mixed incubated and collected using only software control
19. ended before adding to the sample After resuspending do not spin the beads At room temperature add 1 8 volumes of the bead suspension to each sample For example if the fragmented DNA is in a 50 uL volume add 90 uL of the bead suspension Mix thoroughly by pipetting 10 times It may be helpful to use a multichannel pipettor to ensure the incubation times are uniform Incubate at room temperature for 10 minutes Transfer the tubes to the magnet and let stand 5 minutes to completely clear the solution of beads Carefully remove the binding buffer and discard it Leave 15 uL behind to mini mize 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 will impact the amount of DNA carried into ligation 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 and allow to stand for 30 seconds Remove the 70 ethanol wash using a pipette Repeat the 70 ethanol wash two more times for a total of three washes Note With the final wash it is critical to remove as much of the ethanol as pos sible Use at least two 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 Air dry the beads on the magnet for a minimum of 10 minutes Inspect each tube carefully to en
20. ever of the Mondrian SP Workstation forward to the locked position Place the Cartridge Loading Guide on the cartridge or use the guide as a refer ence to identify the correct port for loading Elution Buffer Pipette 50 uL of Elution Buffer into port E5 of the Mondrian SP Cartridge Insert the pipette tip into the port all the way to the bottom of the cartridge When the tip contacts the bottom withdraw the pipette tip slightly to allow space for dis pensing Slowly depress the plunger to dispense the reagent but do not depress the plunger completely blow out as this could introduce bubbles into the car tridge Slowly withdraw the pipette tip from the port performing a final blowout while the tip is within the upper cylinder of the port Note Do NOT add any samples or other reagents to the cartridge at this time Ensure that only Elution Buffer has been loaded Close the lid Select Run on the touch screen menu 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 IV Protocol 13 Ovation SP Ultralow Library Systems 10 Optional Enter run details on the Run Information screen We recommend record ing the serial number of the cartridge on this screen The cartridge serial number can be found on the front of each cartridge for easy reference 11 Select Next
21. her remove and transfer the lower aqueous phase to a new tube or to remove and discard the Filler Fluid oil layer The aqueous phase contains the purified amplified library Store the library at 20 C or proceed immediately to library quantitation V Quantitative and Qualitative Assessment of the Purified Amplified Libraries 22 Ovation SP Ultralow Library Systems A Overview The Quantitative and Qualitative Assessment is used to confirm the average size of the library inserts and calculate library concentration This information is required prior to loading the cluster generation workstation B Recommendations for Library Assessment 1 Runa 1 pL aliquot of the library on the Bioanalyzer High Sensitivity HS DNA Chip The typical distribution of libraries with 200 bp inserts is shown in Figure 4 Note Libraries appear to be closer to 300 bp due to the additional length of ligated adaptors The distribution pattern of the amplified library inserts depends upon the initial frag ment size used to construct the library Following the Ovation SP Ultralow protocol effectively eliminates adaptor dimer formation Any deviation from the protocol may result in dimer formation which will appear as low molecular weight spikes of approxi mately 120 bp on the Bioanalyzer trace Figure 4 Ovation SP Ultralow Library Size Distribution Size distribution of libraries generated from 10 ng of gDNA blue trace or 50 ng of gDNA red trace ru
22. ifacts in the downstream library size analysis These artifacts appear as high molecular weight species during Bioanalyzer or gel analysis Figure 7 This phenomenon is due to the amplification of diverse library molecules that have the same adaptor sequences at their termini As the concentration of library molecules increases during PCR the adaptor ends begin to compete with the PCR primers for hybridization resulting in partially hybridized species Although this may impact PCR efficiency it does not impact library quality for subsequent sequenc ing nor does it affect quantitation by qPCR Figure 7 Fragment distribution on Bioanalyzer Chip 1000 when PCR amplification artifacts are present Fu 7 Marker Marker 604 Red not over amplified so4 Blue over amplified 04 Primers W4 204 104 J i RER 0 M Jarra A i A eae al rr r r r rm 15 30 10 15 2 300 400 500 700 1500 bp If desired performing a single round of PCR in the presence of excess primer will resolve the material to a single peak of the correct library size When quantifying libraries that may have been subject to PCR amplification artifacts use the lower molecular weight peak to estimate library size and qPCR to determine concentration VII Appendix 31 Ovation SP Ultralow Library Systems E Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 What kind of sequencing primers can u
23. igation and avoid adaptor dimer formation The Ovation SP Ultralow Library Systems utilize optimized chemistries to increase the efficiency of blunt end adaptor ligation and minimize the amount of adaptor dimer in the library Does NuGEN provide reagents for performing the fragmentation step of the protocol We recommend using the Covaris instrument as indicated in the Materials section of this user guide NUGEN does not provide the reagents used in the fragmentation steps VII Appendix 32 Ovation SP Ultralow Library Systems Q9 Q10 Q11 Q12 Q13 Which NuGEN amplification system kits can be used to produce cDNA 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 Ovation 3 DGE System Part No 7200 and Ovation Prokaryotic RNA Seq System Part No 9030 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 As a general rule target capture requires a minimum of 500 ng of amplified purified library The amount of purified amplified library generated by the Ovation SP Ultralow Library Systems is not enough for target capture from non multiplexed libraries H
24. is done on a per sample basis and not as a master mix You must prepare and process eight samples on each cartridge We recommend starting sample preparation and reagent preparation once the cartridge has success fully completed QC IV Protocol 14 Ovation SP Ultralow Library Systems Table 2 Sample Solution Preparation volumes listed are for a single sample COMPONENT VOLUME 10 to 100 ng sheared dsDNA in water or low EDTA TE ase Hb Agencourt RNAClean XP beads 4 0 uL Sample Concentration Solution 27 5 uL 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 temperature approximately 23 C for 10 minutes The above recipe is for a single sample Prepare this sample solution for each sample to be processed Mix the samples once again before loading onto the cartridge 2 Ovation SP Ultralow Reagent Master Mix Preparation Prepare Bead Binding Solution Bead Wash Solution and Elution Buffer 1 Remove the Bead Binding Solution Bead Wash Solution and Elution Buffer from the room temperature reagent box 2 Vortex to mix and spin down briefly Leave tubes at room temperature while pre paring the additional master mixes below Prepare DR Multiplex Barcode Adaptors 1 Thaw DR Multiplex Ligation Adaptor Mixes L2V13DR BC1 8 or L2V13DR BC9 16 at room temperature 2 Vortex well
25. le 4 End Repair Master Mix label tube D6 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 IV Protocol E 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 Spin and place the End Repair Enhancer ER3 on ice 2 Prepare master mix in a low binding 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 5 Label the tube D7 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 label tube D7 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 Library Amplification Master Mix DMSO will not completely 1 Thaw Amplification Buffer Mix P1 ver 5 Amplification Primer Mix P2 ver 11 thaw on ice Thaw at room and DMSO P4 at room temperature and vortex to mix well Spin and place the temperature and vortex to a A i mix well Amplification Enzyme Mix P3 ver 4 on ice 2 Prepare Library Amplification Master Mix in a low binding 0 5 mL microcentrifuge tube or a 0 2 mL PCR tube according to the volumes shown in Table 6 Label the tube E2 3 Mix well by
26. ler Fluid x4 2of2 N A N A P01185 Filler Fluid Vial x4 2of2 N A N A S01556 Sample Concentration Solution 2of2 Clear N A S501588 Bead Binding Solution x2 2of2 Clear N A S01589 Bead Wash Solution x2 2of2 Clear N A S01590 Elution Buffer 2of2 Clear N A P01210 aati i ae 2 of 2 N A N A 501698 Agencourt RNAClean XP Beads PP Clear N A separately 7 Components Ovation SP Ultralow Library Systems B Additional Equipment Reagents and Labware Required Materials Equipment 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 0 5 mL and 0 2 mL tubes 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer Qubit 2 0 Fluorometer and dsDNA HS Assay Kit Life Technologies or appropriate fluorometer and accessories for quantitation of fragmented DNA and amplified libraries Reagents Agilent High Sensitivity DNA Kit Agilent Cat 5067 4626 Isopropyl alcohol Low EDTA TE buffer pH 8 0 Affymetrix Cat 75793 Supplies and Labware Nuclease free pipette tips 0 5 mL and 0 2 mL DNase free low bind microcentrifuge tubes e g Sorenson BioScience Inc SafeSeal Microcentrifuge Tubes with Low Binding Polymer Technology 0 65 mL Cat 11300 Eppendorf DNA LoBind consum
27. lex sequencing However the barcodes were carefully chosen for their ability to parse properly and for color balancing and therefore have strict pairing requirements when performing 2 plex multiplexing Users wishing to perform greater than a 2 plex multiplexing must choose a Duplex Set as defined in Tables 10 and 11 combined with any of the remaining barcoded libraries All barcode sequences are separated by an edit distance of three For further details on the barcode design strategy please refer to Faircloth BC Glenn TC 2012 Not All Sequence Tags Are Created Equal Designing and Validating Sequence Identification Tags Robust to Indels PLoS ONE 7 8 e42543 doi 10 1371 journal pone 0042543 Table 7 Barcode sequences for dedicated read DR adaptors used in Ovation SP Ultralow DR Multiplex System 1 8 Part No 8133 LIGATION 6 NT BARCODE BARCODE BARCODE ADAPTOR MIX SEQUENCE AS READ BY PAIRING PAIRING THE SEQUENCER 2 PLEX gt 2 PLEX L2V13DR BC1 AACCAG Duplex Set 1 L2V13DR BC2 TGGTGA One of the duplex L2V13DR BC3 AGTGAG See dats fronith pect column to the left L2V13DR BC4 GCACTA must be used in L2V13DR BC5 ACCTCA combination with Duplex Set 3 any of the other L2V13DR BC6 GTGCTT remaining six indi vidual barcodes L2V13DR BC7 AAGCCT Duplex Set 4 L2V13DR BC8 GTCGTA VII Appendix 25 Ovation SP Ultralow Library Systems Table 8 Barcode sequences for dedicat
28. ltralow Library Systems workflow Mondrian SP Cartridge Step 2 ener 2 E3 E4 ES E6 E7 Make master mix mi DAADA load in reagent ports DS D D7 oo 000060 tep Sample Collection 210 ng fragmented dsDNA 2 H s s bd Adaptors Ovation SP Ultralow ARARA DR Multiplex Systems Single use only Sampie pit Ovation SP Ultralow SSSSCeee Library Systems reagents Step 3 Mondrian SP Workstation Sample concentration performs the following Bii MESOUOE COL Q End repair steps 1 and 2 Add adaptors and ligate _ Be same purcation 3 4 Steps 4 5 Step 4 Performed off Library quantitation Mondrian SP using qPCR and or Workstation Bioanalyzer a Os 6 2 Step 5 Cluster formation and sequencing The entire workflow requires approximately 45 minutes of hands on time to prepare and load reagent master mixes onto the cartridge and retrieve purified amplified libraries No manual bead or gel purification steps are required Starting with as little Ovation SP Ultralow Library Systems 3 Introduction Ovation SP Ultralow Library Systems as 10 ng of fragmented dsDNA the protocol takes approximately 7 hours to complete depending on the number of library amplification cycles performed The final libraries are ready for cluster formation and single read or paired end sequencing In addition to genomic and other double stranded DNA sources the Ovation SP Ultralow Library Sy
29. n on a High Sensitivity DNA Chip FU 140 120 100 20 35 100 200 300 400 600 2000 10380 bp 2 To quantitate the libraries we recommend using a fluorescence based dou ble stranded DNA quantification system such as the Qubit Fluorometer Life Technologies or a gPCR based method such as the KAPA Library Quantification System KAPA Biosystems Yields will vary depending on input and choice of PCR enrichment cycle number Typical yields are 50 100 ng VI Technical Support 23 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 U S 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 VII Appendix 24 Ovation SP Ultralow Library Systems A Sequences of the Barcodes in the Multiplexed Reactions Barcode sequences and multiplex guidelines for adaptors used in Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 can be found in Table 7 and Table 8 respec tively These 6 nucleotide barcode adaptor sequences must be input into the Illumina Sequencing System prior to parsing of the data You may combine anywhere from 2 16 barcoded libraries to allow for a range of multip
30. owever it may be possible to perform target capture on multiplexed libraries where the amount of multiplexed library exceeds 500 ng You may continue to use the Ovation SP Ultralow Library Systems Part No 8033 and 8034 if you wish to perform target capture on the final amplified library without the need to pool libraries prior to capture Target capture of Ovation SP Ultralow or Ovation SP Ultralow libraries requires the use of the Encore Target Capture Module Part No 0332 in addi tion to the Agilent target capture product How can gel purification be eliminated from the workflow and still pre vent adaptor dimer formation The Ovation SP Ultralow Library Systems workflow uses bead based puri fication and efficient primer design thus eliminating the need for gel based purification Can I use the Ovation SP Ultralow Library System with the Mondrian SP Workstation No The Ovation SP Ultralow Library System kit cannot be used on the Mondrian SP Workstation This protocol is compatible only with the Mondrian SP Workstation Make sure to select the Ovation SP Ultralow protocol from the workstation menu when running this kit on the SP Workstation What are the expected yields from the Ovation SP Ultralow Library System Yields will vary depending on the the quantity of DNA input and the num ber of cycles used in PCR amplification Typical yields within the recom mended input and cycle parameters are 50 100 ng as measured by Qui
31. remove the Filler Fluid tube from the SP Cartridge VII Appendix Cartridge Insertion 1 Raise the lid of the Mondrian SP Workstation to reveal the cartridge deck 2 With the cartridge lever in the up position as shown above hold the cartridge level by the sides with the ports facing up and the gold colored electrodes pointing towards the workstation Carefully insert the cartridge into the cartridge deck flush with the surface of the deck allowing the side rails to guide it into place 3 Ifyou encounter any resistance to the cartridge moving into the deck inspect for foreign objects that may be impeding the cartridge and remove them If the cartridge appears to be catching on the edge of the heater bars or Peltier cooling plate you may gently press down on them to allow the cartridge to move forward The heater bars and cooling unit are designed to be slightly flexible in order to engage the cartridge However do not press too hard on the heater bars or Peltier unit as they may become misaligned 28 Ovation SP Ultralow Library Systems VII Appendix 29 Ovation SP Ultralow Library Systems 4 C Confirm that the cartridge is fully inserted into the deck When the cartridge is properly inserted the electrodes are no longer visible and the three guide arrows etched into the front of the deck should be flush with the edge of the cartridge Pull the locking lever forward as illustrated below to engage th
32. rpendicular to the cartridge to make a seal between the cartridge and the pipet tip IV Protocol 21 Ovation SP Ultralow Library Systems 10 Ts Maintaining the seal formed between the pipette tip and the bottom of the cartridge release the plunger Immediately lift the pipette slightly off the bottom of the cartridge to release the seal rapidly drawing Filler Fluid and the sample droplet into the pipette tip Examine the pipette tip to ensure that the appropriately sized droplet is sus pended in the Filler Fluid Dispense the collected fluid including the small sample droplet directly into the 15 uL of water in the collection tube Pipette up and down a few times in the aque ous phase to ensure that all the sample transfers into the water Discard the tip and repeat steps 2 5 to ensure complete collection of the entire sample This is important because occasionally a portion of the sample droplet is left behind in the sample collection port Continue to the next sample collection port and repeat this process until all eight libraries have been collected and placed in separate tubes Remove the cartridge from the workstation and dispose of as appropriate in labo ratory waste Cap the library droplet containing PCR tubes and vortex briefly to mix Spin down briefly to bring the aqueous phase to the bottom of the tube The Filler Fluid oil should remain as a separate layer on top of the aqueous phase Use a pipette to eit
33. se 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 applications 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 We have successfully constructed libraries using fragments ranging from 200 to 500 bp It is possible to generate a library using both smaller and larger frag ments 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 fragmentation such as sonication may also be suitable How does your protocol improve the efficiency of l
34. sssesseceeessesessesseeessesessseeeeas 8 A Input DNA ReQuireMent ic csccscscsscisssscsiecsssecsssecssscestsensvessenateseetanesissssisassssanas 8 B Using Ovation SP Ultralow Library Systems on Illumina NGS Systems 8 GC Amplified Obrany Storag Eesaia E A 9 IV Protocol si cic TT TETS seax ssuuees veyenuesceesdy sbecoes 10 As QVGIVIOW 5 ca descsaussncresiccnsy sed ieena iin E ENE E ANTRENE ERa ia Ueneusbedndvocsevecies 10 B Protocol Notes medard rintainen A EEEE ae Ee E aa Oaie 10 C DNAIFrag mentation nccostisesvescosteeisnvcsssnte E EREE 10 D DNA Concentration after Fragmentation ccceeeceeseesesseeeeeeseeseeseeeserentenes 11 E Cartridge Quality Control Check csssrisisiissssiiieisiisrsieieasinesisenaiin 12 F Protocol for the Ovation SP Ultralow Library Systems on the Mondrian SP CartridG sensisse aa ERa EiS 13 V Quantitative and Qualitative Assessment of the Purified Amplified Libraries 22 A POVEM EW oiaren EE E EENE ATE NE E EN 22 B Recommendations for Library Assessment seeeerreeeneererieeerererrrrerersen 22 VI Technical SUPpOrt sssscsscsssssssssscscssessessossscssossassessansesasccsssnsesssenscaesesesassoasscaneassoeseoss 23 MEAPpENdiX siii aa EE R E EEEE 24 A Sequences of the Barcodes in the Multiplexed Reactions 24 B Mondrian SP Cartridge Handling eccecceescesceeseeeeeeceeeeeeeeeseeeeeeeeneeeeeneeens 25 C Cleaning the Workstation Contact Pins sissies 29 D PGR AmpliticationvArtifacts sx
35. stems have been designed for seamless integration with NUGEN s Ovation WGA FFPE System Part No 6200 Ovation RNA Seq System V2 Part No 7102 Ovation RNA Seq FFPE System Part No 7150 Ovation 3 DGE System Part No 7200 Ovation Prokaryotic RNA Seq System Part No 9030 and Encore Target Capture Module Part No 0332 to enable a complete end to end solution for tran scriptome library construction starting with total RNA The Ovation SP Ultralow DR Multiplex Systems 1 8 Part No 8133 and 9 16 Part No 8134 each provide eight unique dedicated read barcoded adaptors to prepare librar ies for multiplex sequencing Together these two kits enable up to 16 plex sequencing although multiplexing is not required in order to use this kit 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 IIx lle GAII MiSeq HiScan SQ or HiSeq NGS platforms without gel based size selection using 10 100 ng input of dsDNA The Ovation SP Ultralow Library Systems generate libraries ready for quantitation in approximately 7 hours C Quality Control Every lot of the Ovation SP Ultralow DR Multiplex Systems 1 8 and 9 16 undergoes functional testing to meet specifications for library generation performance We recommend the use of control samples when beginning experiments and or using a new
36. sure that all the ethanol has evaporated It is critical that all residual ethanol be removed prior to continuing Remove the tubes from the magnet Add 28 uL 1X TE buffer low EDTA to the dried beads Mix thoroughly to ensure all the beads are resuspended IV Protocol 12 Ovation SP Ultralow Library Systems 14 19 16 E Transfer the tubes to the magnet and let stand for 2 minutes Carefully remove 25 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 let stand briefly on a magnet to minimize bead carryover At this point we recommend quantitating your samples using a Qubit or other high sensitivity dsDNA quantitation system Cartridge Quality Control Check The Mondrian SP Cartridge OC protocol confirms the basic functionality of the Mondrian SP Cartridge prior to use We recommend running this protocol with each cartridge prior to preparing or adding samples and reagents ls We recommend cleaning the contact pins on the deck of the workstation prior to running the Mondrian SP Cartridge QC Protocol The cleaning procedure is detailed in Appendix C On the bench top fill the cartridge with Filler Fluid via the Filler Fluid port accord ing to the instructions in Appendix B Carefully transport the cartridge to the workstation and insert it into the deck Pull the cartridge l
37. t the cartridge to the Mondrian SP Workstation insert the cartridge into the deck pull the locking lever and close the lid Mondrian SP Workstation Initialization Instructions 1 If not already ON locate the workstation ON OFF switch at the back of the work station and turn it to ON Press the On button on the front of the workstation Select Run from the Main Menu touch screen then select the Ovation SP Ultralow protocol with the desired number of cycles 7 9 11 13 or 15 from the Protocol Selection menu Only one protocol may be run per cartridge As a general rule the greater the amount of input DNA the fewer cycles of amplifi cation should be performed For inputs of 50 to 100 ng choose 7 or 9 cycles For inputs of 10 to 50 ng choose 11 or more cycles Note that these are only recommendations The actual number of amplification cycles should be deter mined empirically by the user After you have selected a protocol press Next Follow the instructions on the screen to begin the run When the run is complete approximately 7 hours continue to Library Collection Library Collection from the Mondrian SP Cartridge Place eight low binding 0 2 mL or 0 5 mL microcentrifuge tubes in a rack Add 15 uL of Nuclease free Water green D1 to each tube Use a 100 or 200 uL pipette set to 20 uL Depress the plunger on the pipette and insert the tip all the way to the bottom of the sample collection port pe
38. to mix and spin down briefly Leave tubes at room temperature while preparing the additional master mixes below IV Protocol The ligation buffer is very viscous and must be pipetted 15 slowly to ensure complete aspiration and transfer Ovation SP Ultralow Library Systems Prepare Ligation Master Mix ils Thaw Ligation Buffer Mix L1 ver 5 at room temperature and vortex to mix well Spin and place the Ligation Enzyme Mix L3 ver 4 on ice Prepare master mix in a low binding 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 3 Label the tube D5 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 3 Ligation Master Mix label tube D5 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 End Repair Master Mix ly Thaw End Repair Buffer Mix ER1 ver 5 at room temperature vortex to mix well and spin down briefly Briefly spin down the End Repair Enzyme Mix ER2 ver 4 and place on ice Prepare master mix in a low binding 0 5 mL microcentrifuge tube or 0 2 mL PCR tube according to the volumes shown in Table 4 Label the tube D Mix well by carefully pipetting avoiding the introduction of bubbles Briefly spin down to bring the master mix to the bottom of the tube Tab
39. uld not be used with the Ovation SP Ultralow Library Systems C 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 The fragmented DNA must be concentrated or purified prior to loading on the Mondrian SP Cartridge We have evaluated only Covaris fragmented DNA during the development of the Ovation SP Ultralow Library Systems Other mechanical means of fragmentation such as sonication may also be suitable IV Protocol 11 The purification beads should be removed from 4 C and left at bench top to reach room temperature well before the start of purification Ovation SP Ultralow Library Systems D DNA Concentration after Fragmentation The fragmented DNA may require concentration in order to achieve the desired input This can be done using the Agencourt RNAClean XP bead based purification protocol detailed below provided for your convenience Alternatively you may choose a col umn based purification system that allows small volume elution such as the MinElute Reaction Cleanup Kit QIAGEN Cat 28204 ile 10 11 12 13 Remove the Agencourt RNAClean XP purification beads from 4 C and place on bench top Ensure the beads have completely reached room temperature before proceeding Resuspend the beads by inverting or briefly vortexing the tube Ensure the beads are fully resusp
40. y quantitation and qualification prior to sequencing B 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 Systems e We recommend the routine use of a positive control DNA Especially the first time you set up a reaction using a positive control DNA will allow you to estab lish a baseline of performance and provide the opportunity to become familiar with the protocol e Use the water provided with the kit green 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 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 Buffers adaptors and primers may be thawed at room temperature followed by brief vortexing e Do not warm any enzyme mixes A gentle mix and quick spin down of enzyme mixes is recommended e When placing small 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 Components and reagents from other NUGEN kits sho
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