ThruPLEX DNA-seq Dual Index Quick Protocol
Contents
1. ThruPLEX DNA seq Kit Quick Protocol Dual Indexes For Use With ThruPLEX DNA seq 48D Kit CAT NO R400406 ThruPLEX DNA seg 96D Kit CAT NO R400407 ThruPLEX DNA seq builds on the innovative ThruPLEX chemistry to generate DNA libraries with expanded multiplexing capability and with even greater performance Kits contain either 48 or 96 dual read Illumina compatible indexes pre dispensed and sealed in microplates ThruPLEX DNA seg can be used in DNA seg RNA seq or ChIP seq and offers robust target enrichment performance with all of the leading platforms For more information please visit www rubicongenomics com products ThruPLEX DNA seq For detailed protocol please refer to the ThruPLEX DNA seq Kit Instruction Manual at www rubicongenomics com resources manuals Storage and Handling Store kit at 20 C upon arrival Prior to use transfer enzymes to ice and centrifuge briefly Thaw buffers vortex briefly and centrifuge prior to use Keep all enzymes and buffers on ice until used Technical support Call 734 677 4845 9AM 5 30PM EST or contact support rubicongenomics com Kit Contents Input DNA Sample Requirements ThruPLEX DNA seq Dual Indexes Name Cap Color Pp Requirement i7 Index Sequence i5 Index Sequence Template Preparation Buffer Red Fragmented double stranded DNA Template Preparation Enzyme Read o or cDNA Cells plasma urine other biofluids FFPE tissues fresh tissues frozen tissues Mechanical2. an optimization experiment to identify the appropriate number of PCR cycles needed The table below provides the suggested number of E Fluorescence dyes for detection and optical calibration are ar Shs sige mae PCR cycles at Stage 5 for different input amounts added when monitoring amplification in real time during cycling Hold 2 hour ee Please refer to the Real Time PCR Instrument s user manual for Stage 5 Amplification Guide 8 Remove the plate or tube s from the thermal cycler and centrifuge calibration eye recommendations ne volume Pi deteciion ane DNA Input ng Number of Cycles briefl calibration dyes plus nuclease free water should not exceed 4 uL Ifa y l l regular thermal cycler is used there is no need to add the dyes use Oo 20 OY 6 O 9 Continue to the Library Synthesis Step dial oR nn eas tee water CO a ll Library Synthesis Step E Example EvaGreen Fluorescein dye mix Prepare by mixing i 1 Prepare Library Synthesis Master Mix as described in the table a a Eye a eae Oana 31000 T and 1 500 diluted Fluorescein Calibration Dye Bio Rad below Mix thoroughly with a pipette Keep on ice until used ub Laboratories CAT NO 170 8780 add 2 5 uL of this mx and Library Synthesis Master Mix 1 5 uL of nuclease free water per reaction Cap Color Volume Reaction 3 Remove the seal on the PCR plate or open the tube s E Note Over amplification could result in a higher rate of PCR Libra ry Synthesis B
3. late is used wipe the seal with 70 ethanol and let it dry completely Replace the plastic lid and return the plate to its sleeve and store at 20 C 8 Low level multiplexing Select appropriate dual index combinations that meet Illumina recommended compatibility requirements For more information on multiplexing and index pooling please see plate maps below and refer to the ThruPLEX DNA seq Kit Instruction Manual at www rubicongenomics com resources manuals 9 Index Plate Maps Dual Index Plate 48B or 48D Dual Index Plate 96A or 96D a O em 7 7 D712 The index combination at each well position is indicated by the column i7 and row i5 labels on sSsssa8 sssseossoss 66500008 a60600050608 123 4 5 6 123 4 5 6 7 8 9 11 12 D501 a COCOOOOO OO D501 DOOOOOCO OVDVIOO the plate maps The well colors illustrate one way 0502 s QQOOOO0O OO D502 s OOOOOQO0O000000 to pool dual index combinations for an 8 plex osa c oseeee 2 D503 c paaaaease oo experiment wells sharing the same color should be D504 D D504 D nee A i pooled together For other ways to pool a low plex D506 F 299929 ete a F Oooo Coeg 2 to 16 plex experiment please refer to 0507 c OO OOOO OO 0507 c DOOOOOOOOOO Illumina s TruSeq Sample Preparation Pooling 4 OOO000 OO H DOOOOO00O00 8 D508 D508 Guide Illumina Part 15042173 Rev B 2014 IQ QAM 136 003 RUBICON GENOMICS C Quick Protocol I Template Preparation Step 6 Cent
4. ly sheared enzymatically ATTCAGAA D712 AGCGATAG Nucleic acid FQuick Protocol Cid Type fragmented ChIP DNA low molecular weight cell free DNA A Kit Contents M See table above left B Notes before starting 1 Input DNA sample requirements E See table above middle Please refer to the ThruPLEX DNA seq Kit Instruction Manual for detailed instructions on preparing DNA samples 2 Additional materials and equipment needed Thermal cycler with 50 uL reaction volume capability and heated lid centrifuge PCR tubes or plates PCR plate seals low binding barrier tips fluorescent dyes Agencourt AMPure XP Beckman Coulter CAT NO A63880 80 v v Ethanol 3 Selecting PCR Plates Tubes Select plates tubes that are compatible with the thermal cyclers and or real time PCR instruments used Ensure that there is no evaporation during the process by using proper seal caps during cycling as evaporation may reduce reproducibility 4 Positive and Negative Controls If necessary include a positive control DNA eg Coriell DNA Covaris sheared 200 300 bp and a No Template Control NTC as a negative control in parallel to ensure that the reaction proceeded as expected 5 Preparation of Master Mixes Keep all enzymes and buffers on ice Library Synthesis Master Mix and Library Amplification Master Mix can be prepared during the last 15 minutes of the previous step s cycling protocol and kept on ice un
5. n Master Mix 98 C 67 C lil Library Amplification Step Addition of 4 Mix thoroughly with a pipette Indexes E Note fd solace gage Prin eae 1 one the DIP or the freezer and thaw for 10 min on bench top 72 C l l l l Prior to use centrifuge the DIP to collect the contents at the Librar 98 C 5 Seal the PCR plate using proper sealing film or tightly cap the bottom Wipe foil seal with 70 ethanol and allow to dry Amplifj A 5 me ee 2 P Library Amplification Master Mix as described in th ee _ A repare 1 rary mp wication aster 1x as described in e o oa 4 6 Centrifuge briefly to collect contents to the bottom of each well or table below Mix thoroughly with a pipette Keep on ice until used 6 4c 1 tube Acquire fluorescence data at this step if monitoring in real time Library Amplification Master Mix Cap Color Volume Reaction m Library Amplification Buffer 25 0 uL 7 Place the plate or tube s in a thermal cycler with a heated lid set to Selecti th timal b f lificati les Th 101 C 105 C Perform the Template Preparation Reaction aig pn cae acacia ia eee ee j S number of PCR cycles required at Stage 5 of the Library using the conditions in the table below Library Amplification Enzyme 1 0 uL Template Preparation Reaction Nuclease Free Water plus fluorescent dyes 420 L Amplification Reaction is dependent upon the amount of input DNA and the thermal cycler used We recommend performing
6. q may not be transferred to third parties resold modified for resale or used to manufacture commercial products without prior written approval of Rubicon Genomics Inc ThruPLEX DNA seq is protected by U S Patents 7 803 550 8 071 312 8 399 199 8 728 737 and corresponding foreign patents Additional patents are pending Trademarks ThruPLEX Ilumina TruSeq Illumina Inc EvaGreen Biotium Inc Agencourt AMPure Beckman Coulter Inc l WAYS QAM 36 003 RUBICON GENOMICS
7. rifuge briefly to collect contents to the bottom of each well or 7 Seal the plate or tube s tightly and centrifuge briefly to collect 1 Add 10 uL of DNA sample to each well of a PCR plate or tube If tube contents to the bottom of each well or tube necessary include NTC negative control buffer sample s and 7 Return the plate or tube s to the thermal cycler with a heated lid set 8 Return plate or tube s to the real time PCR thermal cycler thermal positive control samples to 101 C 105 C Perform Library Synthesis Reaction using cycler with a heated lid set to 101 C 105 C Perform Library 2 Depending on the number of reactions prepare the Template the conditions in the table below Amplification Reaction using the cycling conditions from the Preparation Master Mix as described in the table below Mix Library Synthesis Reaction tables below thoroughly with a pipette Keep on ice until used E Caution Ensure that the thermal cycler does not have a Template Preparation Master Mix denaturing step programmed until Stage 3 Cap Color Volume Reaction aS Bcc AA Library Amplification Reaction lempiate Prepetatisn Buffer Red 2l 8 Remove the plate or tube s from the thermal cycler and centrifuge Stage Temperature Time Mumoei of Cycles Template Preparation Enzyme Red briefl Extension amp i Cleavage 3 To each 10 uL sample from step 1 above add 3 uL of the Template 9 Continue to the Library Amplification Step Preparatio
8. til used 6 Indexing Reagents ThruPLEX DNA seq is designed for high throughput applications It is provided with a Dual Index Plate DIP containing either 48 or 96 Illumina compatible dual indexes Each well of the DIP has sufficient volume of Indexing Reagent for a single use and contains a unique combination of Illumina s 8 nucleotide TruSeq HT i5 and i7 index sequences see table above right 7 DIP Handling Instructions The DIP is sealed with pierceable sealing foil and can be frozen and thawed no more than four times Follow the instructions given below to avoid potential index cross contamination Thaw the DIP for 10 min on the bench top prior to use Once thawed briefly centrifuge the plate to collect the contents to the bottom of each well Thoroughly wipe the foil seal with 70 ethanol and allow it to dry completely mM Pierce the seal above each well containing the specific index combination with a clean 200 uL filtered pipet tip discard the tip Use a new pipet tip to collect 5 uL of a specific index combination and add it to the reaction mixture at the Library Amplification Step A multichannel pipette may be used if needed If indexes from the entire plate are not used at the same time low level multiplexing follow the instructions below to avoid contamination Cover any pierced index wells with scientific tape such as VWR General Scientific Tape 0 5 CAT NO 89097 920 to mark the index as used Once the Index P
9. uffer s r duplicates in the library Library Synthesis Enzyme 4 Add 30 uL of Library Amplification Master Mix to each well or tube 9 Remove the plate or tube s from the thermal cycler and centrifuge 2 Remove the seal on the plate or open the tube s 5 Add 5 uL of the appropriate Indexing Reagent from the DIP to briefly 3 Add 2 uL of the Library Synthesis Master Mix to each well or each well or tube E Note At this stage samples can be processed for next generation tube E Note Follow the DIP handling instructions section B 7 of this sequencing NGS reel or stored frozen ica 20 C and 4 Mix thoroughly with a pipette quick protocol to avoid index cross contamination processed later For instructions and recommendations on library l l l Z ie ihi hl a an ee ee pooling purification quantification and sequencing please refer to Note Final volume at this stage is 15 uL i i ab oroughly with a pipette Avoid intro ucing excessive alr fhe ThruPLEX DN A seq Kit i acnmaiecn Manm at 5 Seal the PCR plate using proper sealing film or tightly cap the u CS www rubicongenomics com resources manuals A tube s E Note Final volume at this stage is 50 uL For technical support contact support rubicongenomics com or call 1 734 677 4845 9 AM 5 30 PM EST ThruPLEX DNA seq Kit is for research use only It may not be used for any other purposes including but not limited to use in diagnostics forensics therapeutics or in humans ThruPLEX DNA se
Download Pdf Manuals
Related Search