Procedure & Checklist
Contents
1. Reagent Tube Cap Color Stock Conc Notes DNA ExoVII treated 50 uL DNA Damage Repair 25 X 2 0 uL 1X Mix Total Volume 52 0 uL 1 Mix the reaction well by pipetting or flicking the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 37 C for 20 minutes return the reaction to 4 C for 1 to 5 minutes Repair Ends Use the following table to prepare your reaction then purify the DNA Reagent Tube Cap Color Stock Conc Notes DNA Damage 50 uL Repaired End Repair Mix 20 X 2 5 uL 1X Total Volume 52 5 uL 1 Mix the reaction well by pipetting or flicking the tube 2 Spin down contents of tube with a quick spin in a microfuge 3 Incubate at 25 C for 5 minutes return the reaction to 4 C Page 5 PN 100 286 100 04 STEP VY Purify DNA Notes 1 Add 0 45X volume of AMPure PB beads to the End Repair reaction For detailed instructions on AMPure PB bead purification see the Concentrate DNA section 2 Mix the bead DNA solution thoroughly 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 shaking in a VWR vortex mixer at 2000 rpm for 10 minutes at room temperature 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 superna2. 100 04 Anneal and Bind SMRTbell Templates Use the Binding Calculator to anneal sequencing primer at 0 833 nM concentration and bind polymerase at 0 500 nM concentration You must have the PacBio DNA Polymerase Kit and use LoBind microcentrifuge tubes for this step Before adding the primer to the SMRTbell template the primer must go through a melting step at 80 C This avoids exposing the sample to heat The template and primer mix can then be incubated at 20 C for 30 minutes For polymerase binding incubation at 30 C for 30 minutes is sufficient Instructions for polymerase binding are provided by the calculator For the fields listed below enter or select the following e Average insert size enter the estimated size provided by your Bioanalyzer instrument e Select Magnetic Beads e Small scale reaction e Optional gt Concentration On Plate select Default for 0 025 nM or Custom and enter your chosen loading concentration For more information about using the Binding Calculator see the Pacific Biosciences Template Preparation and Sequencing Guide and QRC Annealing and Binding Recommendations Prepare for MagBead Loading Optimal loading of gt 10 kb templates can be achieved at 0 025 nM on plate concentration A loading titration starting with 0 025 nM on plate concentrations is highly recommended For efficient binding to Magnetic Beads bound complexes at 0 500 nM template concentration must be diluted in the app
3. 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 30 to 60 seconds 13 Elute the DNA off the beads in 50 uL of Elution Buffer Vortex for 1 minute at 2000 rpm 14 The eluted DNA in 50 uL Elution Buffer should be taken into the second 0 45X AMPure PB bead purification step Page 8 PN 100 286 100 04 STEP Purify SMRTbell Templates Second Purification Notes 1 Add 22 5 uL 0 45X volume of AMPure PB beads to the 50 uL of eluted DNA from the first AMPure PB bead purification step above For detailed instructions on AMPure PB bead purification see the Concentrate DNA section 2 Mix the bead DNA solution thoroughly 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 shaking in a VWR vortex mixer at 2000 rpm for 10 minutes at room temperature 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 supernatant and save in another tube Avoid disturbing the bead pellet 8 Wash beads with freshly prepared 70 ethanol 9 Repeat step 8 above 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
4. 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 30 to 60 seconds 13 Elute the DNA off the beads in 100 uL of Elution Buffer Vortex for 1 minute at 2000 rpm 14 Verify your DNA amount and concentration with either a Nanodrop or Qubit quantitation platform reading Page 9 PN 100 286 100 04 STEP Purify SMRTbell Templates Third Purification Notes 1 Add 40 uL or 0 40X of AMPure PB beads to the 100 uL of eluted DNA Note that for 0 40X volumes it is critical to accurately pipet the desired volume of AMPure PB bead solution there is a steep drop off in recovery for concentrations lt 0 40X 2 Mix the bead DNA solution thoroughly 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 shaking in a VWR vortex mixer at 2000 rpm for 10 minutes at room temperature 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 supernatant and save in another tube Avoid disturbing the bead pellet Note It is especially important to save the supernatant for 0 40
5. 2 PACIFIC Procedure amp Checklist BIOSCIENCES Greater Than 10 kb Template Preparation Using AMPure PB Beads Before You Begin To perform this procedure you must have the PacBio Template Prep Kit and have reviewed the User Bulletin Guidelines for Preparing 20 kb SMRTbell Templates This procedure can be used to prepare greater than 10 kb libraries from 5 ug of sheared and concentrated DNA If preparing larger amounts of DNA scale all the reaction volumes proportionally e g if the input amount of DNA is double the amount set forth in this procedure then double all the reaction volumes listed in the tables If a BluePippin size selected system is not available or if the SMRTbell libraries are less than 500 ng an alternative method using AMPure PB beads may be used to filter out most fragments in the 1 kb to 2 kb range A more aggressive approach using 0 40X AMPure PB beads in the final step of the purification process may also be used Sheared and Insert Size Target Insert Size Range Concentrated DNA Ligation Bi hella epair Amount 10 kb to 20 kb gt 10 kb 5 ug Blunt Required Fragment and Concentrate DNA Before shearing your DNA sample be sure to have assessed the quality and purified the sample according to the User Bulletin Guidelines for Preparing 20 kb SMRTbell Templates Use a Covaris g TUBE device to shear your DNA sample Review the recommendations listed in the User Bulletin Guideli
6. X volumes of AMPure PB purification steps In case of low recovery perform a 1X AMPure PB purification step to recover the DNA 8 Wash beads with freshly prepared 70 ethanol 9 Repeat step 8 above 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 30 to 60 seconds 13 Elute the DNA off the beads in 10 uL of Elution Buffer Vortex for 1 minute at 2000 rpm 14 Verify your DNA amount and concentration with either a Nanodrop or Qubit quantitation platform reading For general library yield expect 20 total yield from the Damage Repair input If your yield concentration is below 12 ng uL use the Qubit system for quantitation To estimate your final concentration ng of DNA going into Damage Repair X 0 2 ___ sof Elution Buffer ng uL 15 Perform qualitative and quantitative analysis using a Bioanalyzer instrument Note that typical DNA yield at this point of the process at the end of library preparation is between approximately 5 20 of the total starting DNA amount Page 10 PN 100 286
7. age 11 PN 100 286 100 04
8. e purified DNA where smaller fragments are already eliminated as a result of the shearing process are between 80 100 2 Mix the bead DNA solution thoroughly 3 Quickly spin down the tube for 1 second to collect the beads 4 Allow the DNA to bind to beads by shaking in a VWR vortex mixer at 2000 rpm for 10 minutes at room temperature Note that the bead DNA mixing is critical to yield After vortexing the bead DNA mixture should appear homogenous We recommend using a VWR vortex mixer with a foam microtube attachment see the Guide s Overview section for part numbers If using other instrumentation ensure that the mixing is equally vigorous Failure to thoroughly mix the DNA with the bead reagent will result in inefficient DNA binding and reduced sample recoveries 5 Spin down the tube for 1 second to collect beads 6 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 7 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 recover the DNA 8 Wash beads with freshly prepared 70 ethano
9. l 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 a sufficient 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 9 Repeat step 8 above Page 2 PN 100 286 100 04 STEP JS Concentrate DNA Notes 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 the tube caps open for 30 to 60 seconds 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 uL with preferred mass of at least 5 ug 14 Add the Pacific Biosciences Elution Buffer volume calcula
10. n the range of the specific kit you might be using Note that typical yield at this point of the process i e post shearing and after one 0 45X AMPure PB bead purification is approximately 50 80 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 3 PN 100 286 100 04 ExoVll Treat DNA Note that this is a new step to improve library quality Use the following table to remove single stranded ends from DNA fragments If preparing larger amounts of DNA scale the reaction volumes accordingly i e for 10 ug of DNA scale the total volume to 100 uL Do not exceed 100 ng uL of DNA in the final reaction 1 In a LoBind microcentrifuge tube add the following reagents Reagent Tube Cap Color Stock Conc Volume Final Conc JS Notes Sheared DNA _ yL for 5 0 yg DNA Damage Repair 10 X 5 0 uL 1X Buffer NAD 100 X 0 5 uL 1X ATP high 10 mM 5 0 uL 1 mM dNTP 10 mM 0 5 uL 0 1 mM ExoVll B 10 U uL 1 0 uL 0 2 U L H20 ___uL to adjust to 50 0 uL Total Volume 50 0 uL 2 Mix the reaction well by pipetting or flicking 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 4 PN 100 286 100 04 Repair DNA Damage Use the following table to prepare your reaction
11. nes for Preparing 20 kb SMRTbell Templates The most up to date guidance on how to use the g TUBE device along with recommended centrifuges and centrifugation speeds can be found in the g TUBE device user manual available for download from the Covaris website Depending upon the quality of your sample approximately 20 to 50 sample loss is to be expected as a result of the shearing and concentration process Therefore be sure to have sufficient amounts of starting DNA in order to have at least 5 ug of sheared and concentrated DNA 140 ng uL for the subsequent repair steps Page 1 PN 100 286 100 04 STEP Concentrate DNA Notes 1 Add 0 45X volume of AMPure PB 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 Consistent and efficient recovery of your sample is critical to successful SMRTbell template preparation If using this protocol for the first time we strongly recommend that you process a control sample first Using the DNA shearing methods and subsequent AMPure PB bead purification steps described below you should recover approximately 50 80 of your input DNA by mass Typical yields from pr
12. ntents of tube with a quick spin in a microfuge 2 Incubate at 37 C for 1 hour then return the reaction to 4 C You must proceed with purification after this step Page 7 PN 100 286 100 04 Purify SMRTbell Templates There are 3 purification steps 2 using 0 45X volume of AMPure PB beads and a final purification of 0 40X vol umes of AMPure PB beads STEP Purify SMRTbell Templates Notes 1 Add 0 45X volume of AMPure PB beads to the exonuclease treated reaction For detailed instructions on AMPure PB bead purification see the Concentrate DNA section 2 Mix the bead DNA solution thoroughly 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 shaking in a VWR vortex mixer at 2000 rpm for 10 minutes at room temperature 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 supernatant and save in another tube Avoid disturbing the bead pellet 8 Wash beads with freshly prepared 70 ethanol 9 Repeat step 8 above 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
13. on 1 In a LoBind microcentrifuge tube on ice add the following reagents in the order shown If preparing a Master Mix ensure that the adapter is NOT mixed with the ligase prior to introduction of the inserts Reagent Tube Cap Color eae Volume Final Conc Notes DNA End Repaired 29 0 uL to 30 0 uL Annealed Blunt e 20 uM 1 0 uL 0 5 uM Adapter 20 uM Mix before proceeding Template Prep Buffer O 10 X 4 0 uL 1X ATP low amp 1 mM 2 0 uL 0 05 mM Mix before proceeding Ligase 30 U uL 1 0 uL 0 75 U uL H20 ___uL to adjust to 40 0 pL Total Volume 40 0 uL Note that if size selection is not being performed using a BluePippin system the recommendation is to use 1 uL of adapter as shown above The 10 uL of adapter is not recommended for libraries which are not being size selected using the BluePippin system 2 Mix the reaction well by pipetting or flicking the tube 3 Spin down contents of tube with a quick spin in a microfuge 4 Incubate at 25 C overnight 5 Incubate at 65 C for 10 minutes to inactivate the ligase then return the reaction to 4 C You must proceed with adding exonuclease after this step Add exonuclease to remove failed ligation products Reagent Tube Cap Color Stock Conc JS Volume Ligated DNA 40 uL Mix reaction well by pipetting Exolll 100 0 U uL 1 0 uL ExoVII 10 0 U uL 1 0 uL Total Volume 42 uL 1 Spin down co
14. ropriate ratio of MagBead Binding Buffer and MagBead Wash Buffer Refer to the Calculator for the appropriate dilution recommendations Control Complex Dilution If you will be using the PacBio Control Complex dilute the Control Complex according to the volumes and instructions specified in the Calculator Sequence To prepare for sequencing on the instrument refer to the RS Remote Online Help system or Pacific Biosciences Software Getting Started Guide for more information Follow the touchscreen UI to start your run Note that you must have a DNA Sequencing Kit and SMRT Cells for standard sequencing For Research Use Only Not for use in diagnostic procedures Copyright 2010 2014 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 and to the applicable license terms at http Awww pacificbiosciences com licenses html Pacific Biosciences the Pacific Biosciences logo PacBio SMRT SMRTbell and Iso Seq are trademarks of Pacific Biosciences in the United States and or certain other countries All other trademarks are the sole property of their respective owners P
15. tant and save in another tube Avoid disturbing the bead pellet 8 Wash beads with freshly prepared 70 ethanol 9 Repeat step 8 above 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 30 to 60 seconds 13 Elute the DNA off the beads in 30 uL Elution Buffer Mix until homogenous then vortex for 1 minute at 2000 rpm 14 Optional Verify your DNA amount and concentration using a Nanodrop or Qubit quantitation platform as appropriate 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 The End Repaired DNA can be stored overnight at 4 C or at 20 C for longer duration 17 Actual recovery per uL and total available sample material Page 6 PN 100 286 100 04 Prepare Blunt Ligation Reaction Use the following table to prepare your blunt ligation reacti
16. ted in step 13 above to your beads Thoroughly resuspend beads by vortexing for 1 minute at 2000 rpm If the beads appear over dried or cracked let the Elution Buffer sit on the beads for 2 to 3 minutes then vortex again 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 sample with the working solution The latter part of this dilution factor can be calculated automatically by the Qubit system Discard the beads 15 Perform qualitative and quantitative analysis using a Bioanalyzer instrument Note that the Bioanalyzer instrument has different kits in its offering and the appropriate kit based on insert size should be used Dilute the samples appropriately before loading on the Bioanalyzer chip so that the DNA concentration loaded falls well within the detectable minimum and maximum range of the assay Refer to Agilent Technologies guides for specific information o
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