Unsupported Protocol – Preparing > 30 kb SMRTbell™ Libraries
Contents
1. Evaluate Genomic DNA Quality We highly recommend using BIO RAD CHEF Mapper XA Pulsed Field Electrophoresis for resolution above 50 kb Other systems do not provide good resolution above 50 kb Genomic DNA suitable for preparing gt 30 kb libraries will migrate almost exclusively above 50 kb for example see Figure 2 Sample 1 If significant portion of the gDNA migrates below 50 kb for example see Figure 2 Sample 2 do not proceed with the 30 kb size selection protocol Instead refer to Shared Protocol 20 kb Template Preparation Using BluePippin Size Selection System 15 kb Size Cutoff for recommendations on how to prepare gt 6 kb to gt 15 kb libraries T 2 lt 80kb lt 60kb 20kb a lt 20kb 15kb m Figure 2 Evaluation of gDNA quality by pulsed field gel electrophoresis Lane 1 contains the 1 kb extension DNA ladder and Lane 4 the Bio Rad 5 kb DNA ladder Sample 1 provides an example of high quality high molecular weight gDNA The gDNA of Sample 2 should not be used for the production of a gt 30 kb library Optimize Shearing Conditions and Shear gDNA To ensure sufficient yields of final gt 30 kb libraries input gDNA must be sheared carefully so that the average size of fragmented DNA remains well above the desired size selection cut off The response of individual gDNA samples to recommended shearing parameters may differ and must be determined empirically and evaluated by puls2. adjust gDNA concentration to 140 ng uL with EB and proceed directly to ExoVII treatment below STEP 1 v Concentrate DNA Add 0 45X volume of AMPure PB magnetic beads uL of sample X 0 45X uL of beads Note that the beads must be brought to room temperature and all AMPure PB bead purification steps should be performed at room temperature Before using mix the bead reagent well until the solution appears homogenous Pipette the reagent slowly since the bead mixture is viscous and precise volumes are critical to the purification process Notes Mix bead DNA solution thoroughly by tapping the tube gently Do not pipet to mix Quickly spin down the tube for 1 second to collect the beads Allow the DNA to bind to beads by gentle end over end rotation for 20 minutes at room temperature We recommend using a WWR tube rotator Spin down the tube for 1 second to collect beads Place the tube in a magnetic bead rack until the beads collect to the side of the tube and the solution appears clear The actual time required to collect the beads to the side depends on the volume of beads added With the tube still on the magnetic bead rack slowly pipette off cleared supernatant and save in another tube Avoid disturbing the bead pellet If the DNA is not recovered at the end of this Procedure you can add equal volumes of AMPure PB beads to the saved supernatant and repeat the AMPure PB bead purification steps to
3. gt 40 kb library If the gDNA sample appears under sheared try smaller target shear sizes for example 40 kb for a gt 30 kb library and or a lower DNA concentration until you achieve a similar distribution of fragmented gDNA If the gDNA sample is over sheared try a larger target shear size for example 75 kb for a gt 30 kb library It is also possible to increase or decrease the number of shearing cycles contact Diagenode customer support to enable this option PacBio has not has not developed a tested protocol for this option MR_75kb Input K12 gDNA Input K12 gDNA MR_50kb lt 80kb 60kb lt 40kb 5 2040 ew lt 10kb ee 5kb Figure 3 Evaluation of gDNA shears produced by Megaruptor MR The first lane contains the 1 kb extension DNA ladder and the last the Bio Rad 5 kb DNA ladder MR_50kb and MR_75kb are good shears for a 30 kb size selection Sample g TUBE is oversheared and not appropriate for 30 kb size selection Input K12 gDNA E coli genomic DNA g TUBE genomic DNA sheared with g TUBE MR_50kb 50kb shear size in the Megaruptor software MR_75kb 75kb shear size in the Megaruptor software Page 4 amp Eav CIERIGES Unsupported Protocol Concentrate DNA Using AMPure PB Beads if necessary If the concentration of sheared gDNA is less than 140 ng uL concentrate the sheared gDNA using AMPure PB Bead purification before proceeding If the sheared gDNA concentration is greater than 140 ng uL
4. 30 kb or 40 kb size selection of the SMRTbell templates Note that you must use BluePippin Software v6 20 and the 0 75 DF Marker U1 high pass 30 kb 40 kb v3 run protocol for this procedure A new size standard is required for this protocol Use the U1 marker 1 Prepare up to 5 ug SMRTbell templates in a final volume of 30 uL Elution Buffer for each lane Size selection using this protocol can be aggressive and if not careful recovery may be impacted Itis not recommended to use less than 4 ug per lane Bring the Loading Solution to room temperature then add 10 uL of the Loading Solution to the 30 uL DNA sample For multiple lanes scale volumes proportionally The Loading Solution is viscous so pipet slowly to ensure complete transfer into the DNA sample a Mix by tapping the tube gently do not vortex b Spin briefly to collect the contents at the bottom of the tube Follow the manufacturer s recommendations to set up a run protocol a When setting up the run protocol select the 0 75 DF marker S1 high pass 30 40kb v3 cassette definition file b Using the Range selection mode enter the desired BPstart value of 30000 or 40000 bp A BP End value of 80000 bp should automatically appear Be sure to assign a marker lane Load samples and start the run Be sure to include the U1 marker in the correct lane Typical run times are 10 hours To maximize recovery of eluted DNA wait at least 45 minutes
5. Mix Total Volume 50 0 uL 1 Mix the reaction well by gently tapping the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 37 C for 60 minutes return the reaction to 4 C for 1 to 5 minutes Repair Ends Use the following table to prepare your reaction For more than 5 ug input DNA scale all reaction volumes proportionally Reagent Tube Cap Color Stock Conc Volume Final Conc VA Notes DNA Damage z 52 0 uL Repaired End Repair Mix 20 X 2 5 uL 1X Total Volume 52 5 uL 1 Mix the reaction well by gently tapping the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 25 C for 5 10 minutes return the reaction to 4 C Page 8 amp PACIFIC BIOSCIENCES Purify DNA Using 0 45X AMPure PB Beads STEP Z Purify DNA Unsupported Protocol Notes 1 Add 0 45X volume of AMPure PB beads to the End Repair reaction 2 Mix the bead DNA solution thoroughly by gently tapping the tube 3 Quickly spin down the tube for 1 second to collect the beads Do not pellet beads 4 Allow the DNA to bind to beads by gentle rotation for 20 minutes at room temperature W e recommend using a WR tube rotator 5 Spin down the tube for 1 second to collect beads 6 Place the tube in a magnetic bead rack to collect the beads to the side of the tube 7 Slowly pipette off cleared sup
6. after the run terminates before removing the sample from the elution chamber a Collect the eluate into a 1 5 mL DNA LoBind tube b Wash elution well with 40 uL of Sage Science s 0 1 Tween 20 Wash Solution and add wash to eluted sample Washing the elution well may increase yield 10 20 Page 12 amp EA A AA Unsupported Protocol Purify Size Selected SMRTbell Templates with 0 45X AMPure PB Beads STEP oS Purify DNA Notes 1 Add 0 45X volume of AMPure PB beads to the End Repair reaction 2 Mix the bead DNA solution thoroughly by gently tapping the tube 3 Quickly spin down the tube for 1 second to collect the beads Do not pellet beads 4 Allow the DNA to bind to beads by gentle rotation for 20 minutes at room temperature W e recommend using a WR tube rotator 5 Spin down the tube for 1 second to collect beads 6 Place the tube in a magnetic bead rack to collect the beads to the side of the tube Slowly pipette off cleared supernatant and save in another tube Avoid disturbing the bead pellet 8 W ash beads with freshly prepared 70 ethanol 9 Repeat step 8 10 Remove residual 70 ethanol Remove tube from magnetic bead rack and spin to pellet beads Both the beads and any residual 70 ethanol will be at the bottom of the tube Place the tube back on magnetic bead rack Pipette off any remaining 70 ethanol 11 Check for any remaining droplets in the tube If drople
7. AMPure PB Beads STEP J Purify DNA Add 0 45X volume of AMPure PB beads to the End Repair reaction Unsupported Protocol Notes Mix the bead DNA solution thoroughly by gently tapping the tube Quickly spin down the tube for 1 second to collect the beads Do not pellet beads A OI N Allow the DNA to bind to beads by gentle rotation for 20 minutes at room temperature W e recommend using a WR tube rotator o Spin down the tube for 1 second to collect beads o Place the tube in a magnetic bead rack to collect the beads to the side of the tube Slowly pipette off cleared supernatant and save in another tube Avoid disturbing the bead pellet W ash beads with freshly prepared 70 ethanol Repeat step 8 10 Remove residual 70 ethanol Remove tube from magnetic bead rack and spin to pellet beads Both the beads and any residual 70 ethanol will be at the bottom of the tube Place the tube back on magnetic bead rack Pipette off any remaining 70 ethanol 11 Check for any remaining droplets in the tube If droplets are present repeat step 10 12 Remove the tube from the magnetic bead rack and allow beads to air dry with tube caps open for 60 seconds 13 For up to 10 ug input sheared gDNA elute in 31 uL Elution buffer If you started with more than 5 ug input sheared gDNA scale volume of EB proportionally i e for 20 ug of DNA elute in 62
8. Figure 4 Evaluation of gt 30 kb and gt 40 kb libraries using pulsed field gel electrophoresis The first lane contains the 1 kb extension DNA ladder and the last the Bio Rad 5 kb DNA ladder Samples are as indicated above each lane Anneal and Bind BluePippin Size Selected SMRTbell Templates Using the Binding Calculator compute the molarity of your library by using an average insert size of 30000 bp for gt 30 kb libraries and 40000 bp for gt 40 kb libraries Anneal 20X sequencing primer at a template concentration of 0 833 nM as directed by the Binding Calculator Before adding the primer to the SMRTbell template pre condition the primer by heating to 80 C for 2 minutes then placing immediately on ice Note that if kept on ice during use and stored at 20 C pre conditioned primer may be used multiple times without re heating Add the appropriate amounts of pre conditioned primer and 10X Primer Buffer to the SMRTbell template and incubate at 20 C for at least 30 minutes Bind 10X P6 polymerase at an annealed template concentration of 0 500 nM according to the Binding Calculator For gt 30 and gt 40 kb libraries incubation of the binding reaction at 30 C for 4 hours may modestly improve loading relative to 30 minute binding reactions Prepare for MagBead Loading Optimal loading of gt 30 and gt 40 kb SMRTbell libraries using P6 polymerase can typically be achieved using an on plate concentration of 0 225nM to 0 375 n
9. M We recommend you perform an initial loading titration in this range to determine optimal loading for your sample for example 0 225 nM 0 300 nM and 0 375 nM For efficient binding to Magnetic Beads bound complexes at 0 500 nM concentration must be diluted in the appropriate ratio of MagBead Binding Buffer and MagBead Wash Buffer Follow the Binding Calculator instructions to dilute your sample for MagBead binding Page 16 amp Eav A ANN Unsupported Protocol SMRT Cells for Large Scale Sequencing Projects A loading titration should be performed with each new SMRT Cell lot to determine the optimal on plate sample concentrations If possible large multi SMRT Cell genome projects should be completed with a minimum number of lots to decrease the number of loading titrations When placing an order of SMRT Cells indicate on the Purchase Order to MINIMIZE SMRT Cell lots please use this exact phrase Control Complex Dilution If you will be using the PacBio Control Complex dilute the DNA Control Complex according to the volumes and instructions specified in the Calculator Sequence Note that you must have a DNA Sequencing Kit and SMRT Cells for standard sequencing When sequencing gt 30 and gt 40 kb SMRTbell libraries be sure to choose the correct magnetic bead collection protocol v2 or OCPW indicate a20 000 bp insert size and select stage start when setting up your run protocol We recommend 240 or 360 minute movies fo
10. Unsupported Protocol amp PACIFIC BIOSCIENCES Please note the shared protocols described herein may not have been validated by Pacific Biosciences and are prowded as is and without any warranty Use of these protocols is offered to those customers who understand and accept the associated terms and conditions and wish to take advantage of their potential to help prepare samples for analysis using the PacBio System lf any of these protocols are to be used in a production environment it is the responsibility of the end user to perform the required validation Preparing gt 30 kb SMRTbell Libraries Using the Megaruptor Shearing and BluePippin Size Selection System Before You Begin This document provides recommendations for preparing gt 30 kb size selected SMRTbell libraries from 5 ug of starting sheared genomic DNA gDNA Only high quality high molecular weight gDNA may be used for producing gt 30 kb libraries To ensure success gDNA size and integrity must be verified by pulsed field gel electrophoresis before beginning library preparation Additionally conditions for shearing gDNA to a size that can support producing gt 30 kb libraries must also be determined and verified empirically for each sample Overall yields of gt 30 kb libraries are typically 5 10 For large genome projects we recommend starting this procedure with gt 20 ug of high quality gDNA sample Required Materials Vendor Pulsed Field Gel Electro
11. ction volumes proportionally i e for up to 10 ug of DNA use a 100 uL reaction volume Reagent Tube Cap Color Stock Conc Volume Final Conc Notes Size selected DNA _ pL for 5 0 ug DNA Damage Repair 10 X 5 0 uL 1X Buffer NAD 100 X 0 5 uL 1X ATP high 10 mM 5 0 uL 1mM dNTP e 10 mM 0 5 uL 0 1 mM DNA Damage Repair e 25X 2 0 uL 1X Mix H2O __ pL to adjust to 50 0 uL Total Volume 50 0 uL 2 Mix the reaction well by gently tapping the tube 3 Spin down contents of tube with a quick spin in a microfuge 4 Incubate at 37 C for 60 minutes Page 14 2D Ea yA AA Unsupported Protocol Purify Damage Repaired Size Selected SMRTbell Templates with 0 45X AMPure PB Beads STEP J Purify DNA Notes 1 Add 0 45X volume of AMPure PB beads to the End Repair reaction 2 Mix the bead DNA solution thoroughly by gently tapping the tube 3 Quickly spin down the tube for 1 second to collect the beads Do not pellet beads 4 Allow the DNA to bind to beads by gentle rotation for 20 minutes at room temperature W e recommend using a W R tube rotator 5 Spin down the tube for 1 second to collect beads Place the tube in a magnetic bead rack to collect the beads to the side of the tube Slowly pipette off cleared supernatant and save in another tube Avoid disturbing the bead pellet 8 W ash beads with freshly prepared 70 ethanol 9 R
12. d mass of at least 5 ug 14 Add the Pacific Biosciences Elution Buffer volume calculated in step 13 to your beads Tap the tube with finger gently to mix Do not pipet to mix Elute the DNA by gentle letting the mix stand at room temperature for 2 minutes Spin the tube down to pellet beads then place the tube back on the magnetic bead rack Perform concentration measurements Verify your DNA concentration using a Nanodrop or Qubit quantitation platform If the DNA concentration is estimated to be equal to or below 12 ng uL a Qubit system reading is required When performing a Qubit system reading ensure that your sample is within the range of the Qubit kit you are using For proper concentration calculations incorporate the dilution factor used when diluting your sample to be within range of the Qubit kit and the dilution factor when diluting your sam ple with the working solution The latter part of this dilution factor can be calculated automatically by the Qubit system Discard the beads 15 Perform concentration measurements Measure the DNA concentration using a Qubit fluorometer Using 1 uL of the eluted sample make a 1 10 dilution in EB Use 1 uL of this 1 10 dilution to measure the DNA concentration using a Qubit fluorometer and the dsDNA HS Assay kit according to the manufacturer s recommendations Yield up to this point should be 40 60 The remaining 9 uL of 1 10 dilute
13. d sample may be used for QC by pulsed field gel electrophoresis see example in Figure 4 16 The sheared DNA can be stored for up to 24 hours at 4 C or at 20 C for longer duration 17 Actual recovery per uL and total available sample material Page 6 2D PACIFIC BIOSCIENCES ExoVII Pre treatment of DNA Unsupported Protocol Use the following table to set up a reaction to remove single stranded ends from 5 yg of sheared gDNA at 140 ng uL If starting with more than 5 ug of sheared gDNA scale reaction volumes proportionally i e for 10 ug of DNA scale the total volume to 96 uL Reagent Tube Cap Color Stock Conc Volume Final Conc Notes Sheared DNA 5 ug 36 0 uL DNA Damage Repair 10 X 5 0 pL 1X Buffer NAD 100 X 0 5 uL 1X ATP high 10mM 5 0 pL 1mM dNTP ay 10mM 0 5 uL 0 1mM ExoVII 10 U uL 1 0 uL 0 2 U uL Total Volume 48 0 uL 2 Mix the reaction well by gently tapping the tube 3 Spin down contents of tube with a quick spin in a microfuge 4 Incubate at 37 C for 15 minutes then return the reaction to 4 C Page 7 amp PACIFIC BIOSCIENCES Repair DNA Damage Unsupported Protocol Use the following table to prepare your reaction For more than 5 ug input DNA scale all reaction volumes proportionally Reagent Tube Cap Color Stock Conc Volume Final Conc S Notes DNA ExoVII treated B 48 uL DNA Damage Repair 25 X 2 0 uL 1X
14. ed field gel electrophoresis Test shears are highly recommended Note that for preparing gt 30 kb libraries gDNA may be sheared by using Dagenode s Megaruptor Here we provide initial starting parameters methods as well as strategies for optimization of gDNA shearing if necessary Page 3 PACIFIC 2D BIOSCIENCES Unsupported Protocol Shearing Using Diagenode s Megaruptor For shearing gDNA using Diagenode s Megaruptor generally follow the manufacturer s recommendations 1 Dilute your gDNA in EB to 50 ng L in a volume of 50 uL to 400 uL It is important not to exceed this DNA concentration during Megaruptor shearing or you may clog the hydropore Before shearing remove a 4 uL aliquot for QC un sheared sample 2 To shear gDNA for preparation of a gt 30 kb library choose a target shear size of 50 kb in the Megaruptor software for a gt 40 kb library choose a target shear size of 75 kb For both of these target shear sizes you must use Long Hydropores and have the Long Hydropore option selected in the Megaruptor software 3 Evaluate the distribution of the resulting sheared gDNA by running the un sheared and sheared samples on a BIO RAD CHEF Mapper XA Pulsed Field Electrophoresis Other systems do not provide good resolution above 50 kb Typical results are shown in Figure 3 for a bacterial gJDNA sample The MR_50kb shear shown here was used to prepare a gt 30 kb library and the MR_75kb shear was used to prepare a
15. epeat step 8 10 Remove residual 70 ethanol Remove tube from magnetic bead rack and spin to pellet beads Both the beads and any residual 70 ethanol will be at the bottom of the tube Place the tube back on magnetic bead rack Pipette off any remaining 70 ethanol 11 Check for any remaining droplets in the tube If droplets are present repeat step 10 12 Remove the tube from the magnetic bead rack and allow beads to air dry with tube caps open for 60 seconds 13 For up to 5 yg size selected library elute in 10 uL Elution buffer For more than 5 ug of SMRTbell template scale volume of EB proportionally i e for up to 10 ug of input DNA elute in 20 uL EB Mix by gently tapping the tube elute by lettting it sit for two minutes at room temperature 14 Verify your DNA amount and concentration using a Qubit quantitation platform Using 1 uL of the purified sample make a 1 10 dilution in EB Use 1 uL of this 1 10 dilution to measure the DNA concentration using a Qubit fluorometer and the dsDNA HS Assay kit according to the manufacturer s recommendations The remaining 9 uL of 1 10 diluted sample may be used for QC by pulsed field gel electrophoresis see example in Figure 4 Page 15 amp Ea y CIERIGEE Unsupported Protocol ex x ra Q Q Q g3 o22 2 2 6866655 gt SOO FF V Pog A A_ A 40kb E 40kb gt a gee fs 30kb 20kb ome 10kb _
16. ernatant and save in another tube Avoid disturbing the bead pellet 8 W ash beads with freshly prepared 70 ethanol 9 Repeat step 8 10 Remove residual 70 ethanol Remove tube from magnetic bead rack and spin to pellet beads Both the beads and any residual 70 ethanol will be at the bottom of the tube Place the tube back on magnetic bead rack Pipette off any remaining 70 ethanol 11 Check for any remaining droplets in the tube If droplets are present repeat step 10 12 Remove the tube from the magnetic bead rack and allow beads to air dry with tube caps open for 60 seconds 13 For 5 ug input sheared gDNA elute in 23 uL Elution buffer If you started with more than 5 ug input sheared gDNA scale volume of EB proportionally i e for 10 ug of DNA elute in 46 uL EB Mix by gently tapping the tube Elute by letting it sit at room temperature for 2 minutes 14 Optional Verify your DNA amount and concentration using a Qubit quantitation platform 15 The End Repaired DNA can be stored overnight at 4 C or at 20 C for longer durations 16 Actual recovery per uL and total available sample material End Repaired insert DNA can be stored overnight at 4 C or at 20 C for longer durations Page 9 amp PACI BIOSCIENCES FIC Prepare Blunt Ligation Reaction Unsupported Protocol Use the following table to prepare your reaction adding the components below in the order listed Be sure to mix insert
17. gDNA and adapter BEFORE adding ligase For more than 5 ug input DNA scale all reaction volumes proportionally Reagent Tube Cap Color Stock Volume Final Conc ff Notes Conc DNA End Repaired 23 0 pL Annealed Blunt 20 uM 10 uL 5 uM Adapter 20 uM Finger tap to mix before proceeding Template Prep Buffer O 10X 4 0 uL 1X ATP low amp 1 mM 2 0 uL 0 05 mM Finger tap to mix before proceeding Ligase 30 U uL 1 0 uL 0 75 U L Total Volume 40 0 uL 1 Mix the reaction well by gently tapping the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 25 C or room temperature overnight 4 Incubate at 65 C for 10 minutes to inactivate the ligase then return the reaction to 4 C Exolll VII Digestion to Remove Failed Ligation Products Use the following table to prepare your reaction For more than 5 ug input DNA scale all reaction volumes proportionally Reagent Tube Cap Color Stock Conc Volume Ligated DNA 40 uL Exolll e 100 0 U uL 1 0 uL ExoVII amp 10 0 U uL 1 0 uL Total Volume 42 uL 1 Mix the reaction well by gently tapping the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 37 C for 1 hour then return the reaction to 4 C You must immediately proceed with AMPure PB bead purification after this step Page 10 amp PACIFIC BIOSCIENCES Purify SMRTbell Templates with 0 45X
18. phoresis Pulsed Field Gel Electrophoresis System CHEF Mapper XA Pulsed Field Certified Agarose CHEF DNA Size Standard 5 kb Invitrogen 1 kb DNA extension ladder Shearing Megaruptor Long Hydropores Hydrotubes SMRThell Library SMRTbell Template Prep Kit 1 0 SMRTbell Damage Repair Kit Ampure PB Beads Tube rotator or equivalent Qubit 3 0 Fluorometer and dsDNA HS Assay Kit or equivalent Size Selection BluePippin Size Selection System BluePippin Gel Cassettes Page 1 Bio rad Bio rad Bio rad Life Technologies Diagenode Diagenode Diagenode Pacific Biosciences Pacific Biosciences Pacific Biosciences VWR Life Technologies Life Technologies Sage Science Sage Science 170 3670 162 0137 170 3624 1051 1 012 B06010001 E07010002 C30010018 100 259 100 100 465 900 100 265 900 10136 084 Q33216 Q32854 BLUOOO1 PAC30KB 2D EA A EN Unsupported Protocol Workflow QC gDNA by pulsed field gel electrophoresis Optimize shearing conditions test shears and QC by PFGE Large scale shears and QC by PFGE ExoVII treatment DNA damage repair End repair 0 45x Ampure 7 bead purification ree ligation ones digestion 0 45x Ampure T bead purification Size selection 0 45x Ampure 7 bead purification DNA damage repair l 0 45x Ampure PB bead purification QC Prepare for sequencing Figure 1 Workflow for preparing gt 30 kb SMRTbell libraries Page 2 2D Eav A ANN Unsupported Protocol
19. r gt 30 and gt 40 kb libraries For Research Use Only Not for use in diagnostic procedures Copyright 2015 Pacific Biosciences of California Inc All rights reserved Information in this document is subject to change without notice Pacific Biosciences assumes no responsibility for any errors or omissions in this document Certain notices terms conditions and or use restrictions may pertain to your use of Pacific Biosciences products and or third party products Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale andto the applicable license terms at http www pacificbiosciences com lice nses html Pacific Biosciences the Pacific Biosciences logo PacBio SMRT SMRTbell and Iso Seq are trademarks of Pacific Biosciences BluePippin and SageELF are trademarks of Sage Science Inc NGS go and NGSengine are trademarks of GenDx All other trademarks are the sole property of their respective owners Page 17
20. recover the DNA W ash beads with freshly prepared 70 ethanol Note that 70 ethanol is hygroscopic and should be prepared FRESH to achieve optimal results Also 70 ethanol should be stored in a tightly capped polypropylene tube for no more than 3 days Do not remove the tube from the magnetic rack Use asufficient volume of 70 ethanol to fill the tube 1 5 mL for 1 5 mL tube or 2 mL for 2 mL tube Slowly dispense the 70 ethanol against the side of the tube opposite the beads Let the tube sit for 30 seconds Do not disturb the bead pellet After 30 seconds pipette and discard the 70 ethanol Repeat step 8 10 Remove residual 70 ethanol Remove tube from magnetic bead rack and spin to pellet beads Both the beads and any residual 70 ethanol will be at the bottom of the tube Place the tube back on magnetic bead rack Pipette off any remaining 70 ethanol 11 Check for any remaining droplets in the tube If droplets are present repeat step 10 Page 5 2D Eav A AEN Unsupported Protocol STEP V 12 Concentrate DNA Remove the tube from the magnetic bead rack and allow beads to air dry with the tube caps open for 60 seconds Notes 13 Calculate appropriate volume of Elution Buffer ng X 0 5 ng L uL of Elution Buffer needed The minimum DNA concentration required to proceed to the next step End Repair is 140 ng L with preferre
21. ts are present repeat step 10 12 Remove the tube from the magnetic bead rack and allow beads to air dry with tube caps open for 60 seconds 13 For up to 5 ug input non size selected library gDNA elute in 23 uL Elution buffer If you size selected more than 5 ug of SMRTbell template scale volume of EB proportionally i e for up to 10 yg of input DNA elute in 20 uL EB Mix by gently tapping the tube elute by letting the tube sit for two minutes at room temperature 14 Verify your DNA amount and concentration using a Qubit quantitation platform Using 1 uL of the purified sample make a 1 10 dilution in EB Use 1 uL of this 1 10 dilution to measure the DNA concentration using a Qubit fluorometer and the dsDNA HS Assay kit according to the manufacturer s recommendations Size selection set yield should be 20 40 The remaining 9 uL of 1 10 diluted sample may be used for QC by pulsed field gel electrophoresis 15 Actual recovery per uL and total available sample material AMPure PB bead purified size selected libraries may be stored at 20 C Page 13 2D PACIFIC BIOSCIENCES Repair DNA Damage After Size Selection Unsupported Protocol Using the table below set up a reaction to repair any DNA damage present in SMRTbell templates after gt 30 kb or gt 40 kb size selection For up to 5 pg of DNA use a reaction volume of 50 uL If starting with more than 5 ug of size selected template scale rea
22. uL EB Mix by gently tapping the tube Elute by letting it sit at room temperature for 2 minutes 14 Verify your DNA amount and concentration using a Qubit quantitation platform Measure the DNA concentration using a Qubit fluorometer Using 1 uL of the eluted sample make a 1 10 dilution in EB Use 1 uL of this 1 10 dilution to measure the DNA concentration using a Qubit fluorometer and the dsDNA HS Assay kit according to the manufacturer s recommendations Yield up to this point should be 40 60 The remaining 9 uL of 1 10 diluted sample may be used for QC by pulsed field gel electrophoresis see example in Figure 4 15 Optional Perform qualitative and quantitative analysis using a Bioanalyzer instrument with the DNA 12000 Kit Note that typical yield at this point of the process following End Repair and one 0 45X AMPure PB bead purification is approximately between 80 100 of the total starting material 16 Actual recovery per uL and total available sample material Proceed immediately with size selection after AMPure PB Bead purification of exonuclease treated libraries Samples may be stored at 20 C for short periods at this stage if necessary Page 11 2D Eav A AN Unsupported Protocol Size Selection Using the BluePippin System Follow the instructions in the BluePippin User Manual and User Guides see www sagescience com and the specific recommendations below for gt
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