Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems QUICK REFERENCE Note: For safety and biohazard guidelines, refer to the Safety section in the Fragment Library Preparation Using the AB Library Builder System: 5500 Series SOLiD Systems User Guide (Part no. 4460965). For every chemical, read the SDS and follow the handling instructions. Wear appropriate protective eyewear, clothing, and gloves. Prepare to build the library 1 Quantitate the DNA For accuracy, determine sample DNA concentration using a double-stranded DNA-specific fluorescence assay. Use the Invitrogen Qubit dsdna HS Assay Kit (Invitrogen Part no. Q32851 or Q32854) to measure dsdna concentrations from 10 pg/µl to 100 ng/µl. For samples outside this range, use the Qubit dsdna BR Assay Kit for higher concentrations of DNA (Invitrogen Part no. Q32850 or Q32853) or the Invitrogen Quant-iT PicoGreen dsdna Assay Kit for lower concentrations (Invitrogen Part no. P7589). 2 Shear the DNA with the Covaris S220 System Note: For shearing conditions with the Covaris S2 System, see Shear the DNA with the Covaris S2 System on page 7. a. For each library, mix in a 1.5-mL LoBind Tube: Amount DNA 10 ng 5 µg 1 Low TE Buffer Variable µl Shear Buffer 1.2 µl Total 120 µl b. Prepare the Covaris S2 tank: Fill the tank to level 12. Ensure that the circulated water chiller is set to 2 5 C. Supplement the chiller not the actual water bath with 20% ethylene glycol. Ensure that the water bath temperature is 5 10 C. c. Place a Covaris microtube into the loading station and transfer the mixture to the Covaris microtube.
2 Shear the DNA with the Covaris S220 System (continued) d. Shear the DNA using the appropriate program on the Covaris S220 System: IMPORTANT! Ensure that the bath temperature limit is set at 15 C, and keep the bath temperature to 10 C. Condition Setting 3 Set up the AB Library Builder System Instrument for sizeselected or express fragment library preparation Number of cycles 6 Bath temperature 5 C Bath temperature limit 15 C Mode Water quality testing function Frequency sweeping e. Place the Covaris microtube into the loading station, then transfer the 110 µl of sheared DNA to a new 1.5-mL sample tube provided in the Library Builder Fragment Core Kit for 5500 SOLiD. IMPORTANT! If you are preparing barcoded libraries for multiplexed sequencing, for each sequencing run, use at least one of the following full sets of four barcodes: Barcodes 1 4, 5 8, 9 12, 13 16, 17 20, 21 24, 25 28, 29 32, 33 36, 37 40, 41 44, 45 48, 49 52, 53 56, 57 60, 61 64, 65 68, 69 72, 73 76, 77 80, 81 84, 85 88, 89 92, or 93 96. Use only one of the barcoded-t-0xx adaptors for each ligation reaction, unless < 4 libraries are being barcoded. Use the barcodes according to these conditions: If <4 libraries are prepared for sequencing, then use multiple barcodes per library in equal ratios. For example, for 2 libraries, use 2 barcodes for each library. For 3 libraries, use 4 barcode adaptors for each library for a total of 12 barcodes. If 4 libraries are prepared for sequencing and libraries are split into sets of 4 to use full sets of barcodes, then use one set of barcodes for the remaining libraries (1,2,or 3 libraries). There is no need to use multiple barcodes per library in equal ratios. Off Duty Factor 10% Peak Incident Power Cycles/burst 100 Time 175 Watts 60 seconds a. Thaw the AB Library Builder Cartridges on ice or at room temperature 2 hours or until completely thawed. IMPORTANT! Avoid leaving the cartridges at room temperature for longer than necessary to completely thaw them. Avoid repeated freeze-thawings of unused cartridges. IMPORTANT! Before loading the cartridges into the cartridge rack, ensure that the cartridges are completely thawed, particularly reagents in cartridge wells 2 and 3. 2
3 Set up the AB Library Builder System Instrument for sizeselected or express fragment library preparation (continued) b. Inspect the AB Library Builder Cartridges: Cartridge compartment numbers Volumes 1 1300 µl 2 3 1200 µl 4 Empty 5 6 20 µl 7 85 µl 8 90µL 9 10 20 µl 11: Tube to be added after 5 Reaction Buffer tube prepared 42.5 µl (final volume is 85 µl) 12: Unsealed compartment for beads c. Power OFF the instrument. If necessary, remove an installed protocol card, install the appropriate card, then close the card slot and the instrument door. d. Power ON the instrument, then press START. IMPORTANT! Do not remove the protocol card when the instrument is on. e. Open the instrument door, then remove the tip and tube rack and the cartridge rack and from the instrument. f. Gently tap the cartridge on the laboratory bench until any liquid droplets that might be underneath the foil seal are deposited into the bottom of the well. Ensure that wells 2 and 3 are completely thawed. g. Load 1 cartridge per library into the cartridge rack: Load the reagent cartridges into the cartridge rack by sliding each reagent cartridge along the groove in the direction of the arrow until the reagent cartridge clicks into place. Make sure that the notches in the cartridge align with the notches in the cartridge rack. h. Spin the supplied 5 Reaction Buffer tubes, one tube per sample. i. Calculate the amount of P1-T, barcoded adaptors, and 1 Low TE Buffer needed (see Load adaptors in the cartridges in Fragment Library Preparation Using the AB Library Builder System: 5500 Series SOLiD Systems User Guide. Use an equal amount of each adaptor. j. Add 1 Low TE Buffer, P1 and P2 Adaptors to the 5 Reaction Buffer tubes. Vortex, then pulsespin the tube and place it in open position 11 of the cartridge. k. Thoroughly resuspend Agencourt AMPure XP Reagent, then carefully transfer 500 µl to unsealed position 12 of the cartridge. l. Load the tips and tubes into the tip and tube rack according to the positions of the reagents cartridges in the cartridge rack add load adaptors and Agencourt AMPure Reagent: 1. Row S (fourth row): Load with the sample/elution tubes containing 110 µl of sheared DNA. 2. Row T1 and T2 (second and third rows): Load with AB Library Builder Tips inserted into tip holders. Note: Two tip and tip holder sets are required per sample. 3. Row E (first row): Load with labeled sample/elution tubes, with the caps removed and secured. 3
3 Set up the AB Library Builder System Instrument for sizeselected or express fragment library preparation (continued) m. Insert the loaded cartridge rack into the instrument. IMPORTANT! Before inserting the cartridge rack into AB Library Builder Device, ensure that the cartridges are completely thawed, particularly reagents in cartridge wells 2 and 3. WARNING! Do not touch the surface of the heat block. The temperature of the heat block can reach 95 C. Touching the block can cause burns. n. Insert the loaded tip and tube rack into the instrument with row E in the front: Build the library 1 Run the AB Library Builder System a. Press START to select the AB Library Builder System Kit option, then confirm that you have loaded and inserted the cartridge rack and tip and tube rack correctly, IMPORTANT! For correct operation, make sure that the option and protocol card matches the kit you are using. b. Select the script for the kit you are using, then follow the on-screen prompts. c. (Optional) Scan the sample, elution tube, and sample lane barcodes [refer to the AB Library Builder System User Guide (Part no. 4463421)]. d. Close the door to the AB Library Builder Device, then press START. The screen shows the steps and the approximate run time remaining. Size selection is optional. IMPORTANT! Do not open the door during a protocol run. To pause a run press Stop once. To cancel a run press Stop twice. e. At the end of the run, press to return to the Main menu, then open the instrument door, and remove the racks. Confirm that they are properly labeled, then cap the elution tubes containing the library in 100 µl. f. If the library has a brown tint, place each tube in a DynaMag -2 magnetic rack for at least 1 minute until the solution is clear of brown tint when viewed at an angle; then transfer the supernatant to a new tube. g. Properly store the elution tubes and properly dispose of reagent cartridges, tips, and tubes. h. Close the instrument door. No cooling period is required between runs. i. Clean the tip and tube rack and set up for a new run. 4
2 Set up for the next run Clean the tip and tube rack as needed and set up for the next run. Nick-translate and (optional) amplify the library 1 Nick-translate; or nicktranslate and amplify the library Prepare the reaction, then nick-translate the library Note: To nick-translate and amplify the library, proceed to (Optional) Prepare the reaction, then nick-translate and amplify the library below. 1. In a new 1.5-mL LoBind Tube, combine for each library: Volume Platinum PCR Amplification Mix 400 µl Library 100 µl Total 500 µl 2. Vortex the reaction for 5 seconds, then pulse-spin. 3. Distribute 125-µL aliquots of combined library and PCR master mix between 4, 0.2-mL PCR tubes. 4. Incubate the library at 72 C for 20 minutes. 5. Purify the DNA using the Agencourt AMPure XP Reagent (see Purify the DNA on page 7). 6. Proceed to Quantitate the DNA on page 6. (Optional) Prepare the reaction, then nick-translate and amplify the library 1. In a new 1.5-mL LoBind Tube, combine for a PCR master mix: Volume per amplification Master mix for N libraries Platinum PCR Amplification Mix 380 µl 380 µl (1.1 N) Library PCR Primer 1, 50 µm 10 µl 10 µl (1.1 N) Library PCR Primer 2, 50 µm 10 µl 10 µl (1.1 N) Total 400 µl 400 µl (1.1 N) 2. Transfer 400 µl of the PCR master mix to each library. Each library is 100 µl in an elution tube so that the total volume of the mix is 500 µl. 3. Vortex the reaction for 5 seconds, then pulse-spin. 4. Distribute 125-µL aliquots of combined library and PCR master mix between four, 0.2-mL PCR tubes. IMPORTANT! The current protocol is optimized for maximum yield from input DNA. In many cases, library amplification is not needed. Quantitate the library to assess the need to amplify it. If library amplification is needed, minimize the number of cycles, based on the amount of starting input DNA. Use minimal cycling to avoid over-amplification and production of redundant molecules. 5
5. Determine the number of PCR cycles: Starting amount of DNA Number of cycles 10 100 ng 10 cycles 100 ng 1 µg 6 8 cycles 1 2 µg 4 6 cycles 2 5 µg 3 6 cycles IMPORTANT! Minimize the number of PCR cycles to avoid over-amplification and redundant molecules. Base the number of cycles on the amount of starting input DNA. 6. Run the PCR for each 125-µL aliquot: Stage Step Temp Time Holding Nick translation 72 C 20 min Holding Denature 95 C 5 min Cycling Denature 95 C 15 sec Anneal 62 C 15 sec Extend 70 C 1min Holding Extend 70 C 5 min Holding 4 C 7. Purify the DNA using the Agencourt AMPure XP Reagent (see Purify the DNA on page 7). 2 Quantitate the DNA Quantitate the DNA by using the Qubit dsdna HS Assay Kit, the NanoDrop ND-1000 Spectrophotometer, the Agilent Technologies 2100 Bioanalyzer, and/or by qpcr. 3 4 Check the size distribution of the library (Optional) Pool equal molar barcoded libraries of similar size Use 1 µl of sample in the Agilent Technologies 2100 Bioanalyzer. If you see the expected size distribution, proceed directly to emulsion PCR [refer to the SOLiD EZ Bead Emulsifier Getting Started Guide (Part no. 4441486)]. If you do not see the expected size distribution, troubleshoot or contact your Life Technologies Applications Specialist. IMPORTANT! If you are working with libraries of dissimilar sizes or unequal library representation is not acceptable, then do not pool the libraries until after gel purification to avoid library bias. a. Quantitate the libraries to be pooled by qpcr [refer to the Applied Biosystems SOLiD Library TaqMan Quantitation Kit protocol (Invitrogen Part no. A12120)]. b. Mix together equal molar amounts of each barcoded library of similar size in an appropriately sized LoBind Tube. Vortex the tube. c. (Optional) Size select then pool the libraries (refer to Fragment Library Preparation Using the AB Library Builder System: 5500 Series SOLiD Systems User Guide). 6
Supplementary procedures Shear the DNA with the Covaris S2 System 1. For each library, mix in a 1.5-mL LoBind Tube: Amount DNA 10 ng 5 µg 1 Low TE Buffer Variable µl Shear Buffer 1.2 µl Total 120 µl 2. Prepare the Covaris S2 tank: Fill the tank to level 12. Ensure that the circulated water chiller is set to 2 5 C. Supplement the chiller not the actual water bath with 20% ethylene glycol. Ensure that the water bath temperature is 5 10 C. 3. Place a Covaris microtube into the loading station and transfer the mixture to the Covaris microtube. 4. Shear the DNA using the appropriate program on the Covaris S2 System: IMPORTANT! Ensure that the bath temperature limit is set at 15 C, and keep the bath temperature to 10 C. Condition Setting Number of cycles 6 Bath temperature 5 C Bath temperature limit 15 C Mode Water quality testing function 5. Place the Covaris microtube into the loading station, then transfer the 110 µl of sheared DNA to a new 1.5-mL sample tube provided in the Library Builder Fragment Core Kit for 5500 SOLiD. Purify the DNA 1. Resuspend the Agencourt AMPure XP Reagent and allow the mixture to come to room temperature (~30 minutes). 2. Prepare 70% ethanol for N number of libraries: Frequency sweeping Off Duty cycle 10% Intensity 5 Cycles/burst 100 Time 60 seconds Nuclease-Free Water Ethanol, Absolute Total Volume 600 µl N 1400 µl N 2000 µl N 7
3. For every amplified library, label a new 1.5-ml LoBind Tube. 4. Combine each set of the identical 4 PCR reactions (125 µl) to the appropriately labeled 1.5-mL LoBind Tube. The total combined volume of the amplified library is 500 µl. 5. Bind the DNA to the resuspended, ambient Agencourt AMPure XP Reagent: a. For each library, prepare the bead suspension: Volume Amplified library 500 µl Agencourt AMPure XP Reagent 750 µl Equal to 1.5 volumes of sample reaction. b. Vortex the beads for 10 seconds, then pulse-spin. c. Incubate the mixture at room temperature (20 25 C) for 5 minutes. d. Place each tube in a DynaMag -2 magnetic rack for at least 3 minutes until the solution is clear of brown tint when viewed at an angle; then, remove and discard the supernatant. 6. Wash the DNA 2 times. For each wash: a. Without removing the tube from the magnet, add 750 µl of freshly prepared 70% ethanol and incubate for 30 seconds. Do not disturb the pellet. b. Aspirate and discard ethanol. 7. Remove the tube from the DynaMag -2 magnetic rack, pulse-spin the tube, return the tube to the magnetic rack, then remove and discard the supernatant with a 20-µL pipettor. 8. Open each tube, then dry the beads at room temperature (20 25 C) for 5minutes. 9. Elute the DNA: a. Remove each tube from the DynaMag -2 magnetic rack, then add the appropriate volume of Low TE Buffer directly to the pellet to disperse the beads. b. Vortex the beads for 10 seconds, then pulse-spin. c. Incubate the beads for 2 minutes at room temperature. d. Place the tube in a magnetic rack for at least 1 minute until the solution clears. e. Transfer the supernatant containing the amplified library to a new 1.5-mL LoBind Tube. 8
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For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use. NOTICE TO PURCHASER: PLEASE REFER TO THE APPLIED BIOSYSTEMS LIBRARY PREPARATION FOR THE AB LIBRARY BUILDER TM SYSTEM 5500 SERIES SOLiD TM SYSTEMS USER GUIDE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Bioanalyzer is a trademark of Agilent Technologies Inc. Ampure is a registered trademark of Beckman Coulter, Inc. Covaris is a registered trademark of Covaris, Inc. NanoDrop is a registered trademark of NanoDrop Technologies. TaqMan is a registered trademark of Roche Molecular Systems, Inc. Copyright 2011, Life Technologies Corporation. All rights reserved. Part Number 4461307 Rev. A 03/2011 Headquarters 5791 Van Allen Way Carlsbad, CA 92008 USA Phone 760.603.7200 www.lifetechnologies.com Technical Resources and Support For the latest technical resources and support information for all locations, please refer to our Web site at www.appliedbiosystems.com