as low as 100 ng of gDNA
Contents
1. E PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Please note the shared protocols described herein may not have been validated by Pacific Biosciences and are provided 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 If 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 10 kb to 20 kb Template Preparation and Sequencing with Low Input DNA Before You Begin To perform this procedure you must have the PacBio e DNA Template Prep Kit e DNA Polymerase Binding Kit e MagBead Kit e DNA Sequencing Kit DNA Control Complex SMRT Cells for standard sequencing This procedure can be used to prepare 10 20 kb libraries from 50 ng up to 200 ng of sheared and concentrated DNA or at least 100 ng into shearing Note for input amounts between 200 ng and 1 ug the standard 10 kb library prep protocol may be used Sheared and DNAD Insert Size Target Insert Size Range Concentrated DNA Ligation aman Amount Repair 10 to 20 kb 8 kb to 22 kb 50 to 200 ng Blunt Required Fragment and Concentrate DNA Use a Covaris g TUBE device to shear your DNA sample following the g TUBE user manual available for download from the Covaris w2. Vortex for 1 2 minutes at 2000 rpm Spin the tube down to pellet beads then place the tube back on the magnetic bead rack Carefully collect the eluted sample Discard the beads 14 Proceed to the next step if possible If necessary store at 20 C to continue later Page 5 E PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Prepare Blunt Ligation Reaction Use the following table to prepare your blunt ligation reaction 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 Add the adapter to the well with the DNA All other components including the ligase should be added to the Master Mix Stock Reagent Tube Cap Color Volume Final Conc Notes Conc DNA End Repaired 32 ul Annealed Blunt Q 20 uM 1 0 uL 0 5 uM Adapter 20 uM Mix before proceeding 10 X 4 0 uL 1X Template Prep Buffer Mix before proceeding Ligase 30 U L 1 0 uL 0 75 U uL O ATP low e 1 mM 2 0 uL 0 05 mM Total Volume 40 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 25 C for 45 minutes 5 Incubate at 65 C for 10 minutes to inactivate the ligase then return the reaction to 4 C You must proceed with a
3. dding exonucleases after this step Add exonucleases to remove failed ligation products Reagent Tube Cap Color Stock Conc JS Volume Ligated DNA 40 uL Mix reaction well by pipetting Exolll oO 100 0 U L 0 5 uL ExoVII 10 0 U uL 0 5 uL Total Volume 41 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 45 minutes then return the reaction to 4 C Do not exceed 1 hour incubation time You must proceed with purification after this step Page 6 E PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Purify SMRTbell Templates There are 2 final purification steps The first uses 0 5X volumes of AMPure PB beads followed by purification with 0 45X volumes of AMPure PB beads STEP f Purify SMRTbell Templates First Purification Notes 1 Add 0 5X volume of AMPure PB beads to the exonuclease treated reaction For detailed instructions on AMPure PB bead purification see the Concentrate DNA section 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 S
4. ds 14 Proceed to the next step if possible If necessary store at 20 C to continue later Page 3 E PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Repair DNA Damage Use the following table to repair any DNA damage 1 In aLoBind microcentrifuge tube add the reagents below Note premix damage repair buffer NAD ATP high and dNTPs if you are preparing more than 1 sample Reagent Tube Cap Color Stock Conc Volume Final Conc Yi Notes Sheared DNA 37 uL DNA Damage Repair oO 10 X 5 0 uL 1X Buffer NAD 100 X 0 5 uL 1X ATP high e 10 mM 5 0 uL 1 mM dNTP g 10 mM 0 5 uL 0 1 mM DNA Damage Repair e 2 0 uL Mix Total Volume 50 0 uL To determine the correct amount of H O to add use your actual DNA amount noted in the Notes column 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 20 minutes then return the reaction to 4 C for 1 minute Repair Ends Use the following table to prepare your reaction then purify the DNA Reagent Tube Cap Color Stock Conc Final Conc Notes DNA Damage Repaired End Repair Mix a 20 X 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 25 C for 5 minutes no longer return the reac
5. ebsite with one change reduce the sample volume from 150 uL to 50 uL Note After the first spin make sure that all of the sample has passed into the lower chamber If any sample remains re spin If necessary add 10 uL EB or TE to the upper chamber flick the g TUBE or pipette up and down several times and spin again 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 Page 1 PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol STEP JS Concentrate DNA Notes 1 Add 0 5X volume of AMPure PB magnetic beads uL of sample X 0 5X 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 pre purified DNA where smaller fragments are already e
6. ing day The yield will decrease with longer times between runs For Research Use Only Not for use in diagnostic procedures Copyright 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 www pacificbiosciences com licenses html Pacific Biosciences the Pacific Biosciences logo PacBio SMR 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
7. liminated as a result of the shearing process are between 80 100 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 mixing 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 mixing 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 ethanol Note that 70 ethanol is hygroscopic and should be
8. lowly 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 and dry the bead pellet 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 50 uL of Elution Buffer Mix for 10 minute at 2000 rpm Mix until homogeneous Vortex for 1 2 minutes at 2000 rpm Spin the tube down to pellet beads then place the tube back on the magnetic bead rack Carefully collect the eluted sample Discard the beads Page 7 E PACIFIC wy BIOSCIENCES PacBio SampleNet Shared Protocol STEP Yf Purify SMRTbell Templates Second Purification Notes 1 Add 0 45x volume of AMPure PB beads to the 50 uL of eluted DNA 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 a
9. nder Optional below If this number is lt 8 0 nM see PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Optional Concentration On Plate Use Default 0 025 nM Custom 0 025 nM DNA Control Complex Ratio to Template Use Default 1 Custom 0 Polymerase Template Ratio Use Default 10 9 Custom Ces D as __ Increase this number as required until the polymerase dilution is at least 8 0 nM see below Dilutions Step6 Prepare dilutions of polymerase 10000bp Polymerase Dilution 18Pa SA DNA Polymerase P5 1 5uL 1600 nM Binding Buffer v3 292 1 uL Total Volume 293 6 uL Final Concentratio 5 Use the entire complex to sequence the number of SMRT Cells recommended by the calculator in one run 6 If desired the bead bound complex from sample plate can be used the next day by doing the following Pool the remaining beads from multiple SMRT Cells Add the Bead Binding Buffer to bring the volume to 19 uL for 1 SMRT Cell or the required volume for the closest number of SMRT Cells For example if 3 cells were run and the remaining pooled volume is 26 uL add 2 uL Bead Binding Buffer to bring the volume to 28 uL for 2 SMRT Cells The required volume for any number of SMRT Cells may be determined with the binding calculator The expected yield from reused beads depends on the amount of Bead Binding buffer added If none is needed it may be close to the original yield if used the follow
10. or P5 polymerase enter 0 040 nM 3 Under Annealing pre mix 10x Primer Buffer and Diluted Sequencing Primer at higher volumes to eliminate small volume pipetting Optional Concentration On Plate Use Default 0 015 nM Custom DNA Control Complex Ratio to Template Use Default 1 Custom 0 Polymerase Template Ratio Use Default 10 Custom 0 Annealing Step 2 Dilute the Sequencing Primer from 5000 nM to 150 nM in Elution Buffer Sequencing Primerv2 1uL Elution Buffer 32 3 uL Total Volume 33 3 uL Step 3 In 0 2mL tubes add the appropriate amount of reagents in the order listed below F a Preparea 10x pre mix by Voume RPO DE combining 9 0 uL 10x Primer 10x Primer Buffer lt lt i Sample Voume S Buffer and 1 3 uL Diluted Diluted Sequencing Sequencing Primer Primer 7 b Add 1 03 uL pre mix to the Total Volume 9uL Final Concentration 0 109 nM sample Step 4 Incubate 80C for 2 minutes then ramp temperature to 25C at a rate of 0 1C second Step 5 Transfer to 4C location for immediate use or store at 20C 4 Dilutions If polymerase is diluted to lt 8 0 nM increase the polymerase template ratio until 8 0 nM final concentration is obtained or simply dilute polymerase 1 200 in Binding Buffer v3 Dilutions Step6 Prepare dilutions of polymerase 10000bp Polymerase Dilution 18Pa SA DNA Polymerase P5 1 5uL 1600 nM Binding Buffer v3 365 5 uL Total Volume 367 uL Fini Concentration lt recommendation u
11. 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 PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol STEP Concentrate DNA Notes 10 Remove residual 70 ethanol and dry the bead pellet 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 Add 37 uL of Pacific Biosciences Elution Buffer to the beads to elute the DNA Mix until homogeneous Vortex for 1 2 minutes at 2000 rpm Spin the tube down to pellet beads then place the tube back on the magnetic bead rack Carefully collect the eluted sample Discard the bea
12. t have a PacBio DNA Polymerase Binding Kit for this step To anneal sequencing primer and bind polymerase to SMRTbell templates follow the Calculator recommendations with the following set up Under Edit Sample enter the Volume to Use and DNA Concentration measured or estimated assuming 10 library yield Then select the following Magnetic Beads Yes Preparation Protocol Small scale DNA Control Complex No Non standard Yes I aT M Oe 1 eet ice s Sample List New Sample low input test 10000 bp 0 8 ng uL Version 2 1 0 0 Edit Sample saved Delete Step 1 Enter sample information Sample Name low input test Mag bead P4 Small Non Compute standard Volume to Use uL Max of SMRT Cells 3 of SMRT Celis 0 Loading Titration 0 0 Warning Pipetting 0 0 nM Eom volumes will be Details difficult DNA Concentration ng uL Insert Size 10000 bp Magnetic Beads A No Conversion Calculator Binding Kit c2 0 0 ng uL at Ti 2000 base pairs ps equals 0 nM PS Preparation Protocol Small scale Large scale 2000 base pairs at Long Term Storage Yes 0 0 nM No Sa DNA Control Complex Yes Complex Reuse Yes No Non standard N 5 Note the suggested number Max of SMRT ces C 3 o of SMRT Cells Available Volume 0 uL PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol 2 Under Optional enter a Custom Concentration on Plate For P4 polymerase enter 0 025 nM F
13. t 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 and dry the bead pellet 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 8 10 uL of Elution Buffer Mix until homogeneous Vortex for 1 2 minutes at 2000 rpm Spin the tube down to pellet beads then place the tube back on the magnetic bead rack Carefully collect the eluted sample Discard the beads 14 Check quantitation with the Qubit dsDNA HS Assay Kit If there is too little sample estimate concentration based on 10 yield of input amount into damage repair Page 8 2D PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol Anneal and Bind SMRTbell Templates You mus
14. tion to 4 C Page 4 PACIFIC BIOSCIENCES PacBio SampleNet Shared Protocol STEP oY Purify DNA Notes 1 Add 0 5X 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 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 and dry the bead pellet 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 32 33 uL Elution Buffer Mix until homogeneous
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