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1. 10 7 5 8 2 10 1 11 0 2 14 0 05 16 Note Over amplification could result in a higher rate of PCR duplicates in the library 9 Remove the plate or tube s from the thermal cycler and centrifuge briefly Note At this stage samples can be processed for next generation sequencing NGS immediately or stored frozen at 20 C and processed later For instructions and recommendations on library pooling purification quantification and sequencing please refer to the MicroPlex Library Preparation v2 kit Instruction Manual at www diagenode com MicroPlex Library Preparation kit v2 is intended 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 MicroPlex Library Preparation v2 may not be transferred to third parties resold modified for resale or used to manufacture commercial products without prior written approval of Diagenode sa The 8nt index sequences were developed by the Wellcome Trust Sanger Institute in Cambridge UK additional information can be found in Nature Methods 7 111 118 2010 Illumina is a registered trademark of Illumina Inc VAD MicroPlex Library Preparation Kit v2 x12 12 indices and MicroPlex Library Preparation Kit x48 48 indices contains ThruPLEX technology developed and manufactured RUBICON GENOMICS by Rubicon Genomics Inc Ann Arbor Michigan USA and covered by US Patent 7 803 5502. sequence see table below The SIP is sealed with pierceable sealing foil each well has sufficient volume for a single use e MicroPlex Library Preparation kit v2 x12 12 indices are provided with 12 Indexing Reagents pre dispensed in tubes They have sufficient reagents for up to 8 uses and contain 8 nucleotide Sanger indexes see table below that share the same sequences in the first 6 bases as the Illumina TruSeq LT indexes AD001 through ADO12 7 Single Index Plate SIP Handling Instructions Follow the instructions given below to avoid index cross contamination e Thaw the SIP 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 e Pierce the seal above each well containing the specific index combination with a clean 200 uL filtered pipet tip discard the tip e 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 MiSeq only see section B 8 below follow the instructions below to avoid contamination e 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 e Once the Index Plate is
3. EP1924704 and US and international patents pending DIAGENODE S A BELGIUM EUROPE LIEGE SCIENCE PARK Rue Bois Saint Jean 3 4102 Seraing Belgium Tel 32 4 364 20 50 Fax 32 4 364 20 51 orders diagenode com info diagenode com DIAGENODE HEADQUARTERS DIAGENODE INC USA NORTH AMERICA 400 Morris Avenue Suite 101 Denville NJ 07834 Tel 1 862 209 4680 Fax 1 862 209 4681 orders na diagenode com info na diagenode com FOR A COMPLETE LISTING OF DIAGENODE S INTERNATIONAL DISTRIBUTORS VISIT http www diagenode com company distributors php For rest of the world please contact Diagenode sa www diagenode com 2015 Diagenode Inc All rights reserved The content of this document cannot be reproduced without prior permission of the authors QG Microplex 02_15
4. TG D9 TATGCCAG A10 TAGCTTGT B10 TGATACG C10 TAACGCTG D10 TCAGATTC A11 GGCTACAG B11 GTGCTACC C11 TGGTATG D11 TACTAGTC A12 CTTGTACT B12 GGTTGGAC C12 GAACTGG D12 TTCAGCTC C Quick protocol l Template Preparation Step dh Add 10 uL of DNA sample to each well of a PCR plate or tube If necessary include NTC negative control buffer sample s and positive control samples Depending on the number of reactions prepare the Template Preparation Master Mix as described in the table below Mix thoroughly with a pipette Keep on ice until used Template Preparation Master Mix Component Cap Color Volume Reaction Template Preparation Buffer Red 2L Template Preparation Enzyme Red Tul To each 10 uL sample from step 1 above add 3 uL of the Template Preparation Master Mix Mix thoroughly with a pipette Note Final volume at this stage will be 13 uL Seal the PCR plate using proper sealing film or tightly cap the tubels Centrifuge briefly to collect contents to the bottom of each well or tube 7 Place the plate or tube s in a thermal cycler with a heated lid set to 101 C 105 C Perform the Template Preparation Reaction using the conditions in the table below Template Preparation Reaction Temperature Time 22 C 25 min 55 C 20 min 4 C Hold lt 2 hours 8 Remove the plate or tubels f
5. diagend te Innovating Epigenetic Solutions QUICK GUIDE MicroPlex Library Preparation kit v2 For use with MicroPlex Library Preparation kit v2 x12 12 indices Cat No C05010012 MicroPlex Library Preparation kit v2 x48 12 indices Cat No C05010013 MicroPlex Library Preparation kit v2 x48 48 indices Cat No C05010014 MicroPlex Library Preparation v2 builds on the innovative MicroPlex chemistry to generate DNA libraries with expanded multiplexing capability and with even greater performance kits contain either 12 or 48 single read Illumina compatible indexes MicroPlex v2 can be used in DNA seq RNA seq or ChIP seq and offers robust target enrichment performance with all of the leading platforms For detailed protocol please refer to the MicroPlex Library Preparation kit v2 Instruction Manual at www diagenode com 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 A Kit contents ee oe 1 Input DNA sample requirements please refer to the MicroPlex Library Preparation kit v2 Instruction Manual for detailed instructions on preparing DNA samples a Fragmented double stranded DNA or cDNA Cells plasma urine other biofluids FFPE tissues fresh Source tissues frozen tissues Tie Mechanically sheared enzymatically fragmented low yP
6. horoughly with a pipette Avoid introducing excessive air bubbles Note Final volume at this stage is 50 uL Seal the plate or tube s tightly and centrifuge briefly to collect contents to the bottom of each well or tube Return plate or tube s to the real time PCR thermal cycler thermal cycler with a heated lid set to 101 C 105 C Perform Library Amplification Reaction using the cycling conditions from the tables below Caution Ensure that the thermal cycler does not have a denaturing step programmed until Stage 3 Library Amplification Reaction Stage 7 Time Number of Cycles 1 72 C 3 min 1 Extension amp Cleavage 2 85 C 2 min 1 Denaturation 3 98 C 2 min 1 98 C 20s Addition of Indexes 4 67 C 20s 4 72 C 40s r AS 98 C 20s 5 to16 see table Library Amplification 5 72 C 50s below 6 4 C Hold 1 Acquire fluorescence data at this step if monitoring in real time Selecting the optimal number of amplification cycles The number of PCR cycles required at Stage 5 of the Library Amplification Reaction is dependent upon the amount of input DNA and the thermal cycler used We recommend performing an optimization experiment to identify the appropriate number of PCR cycles needed The table below provides the suggested number of PCR cycles at Stage 5 for different input amounts Stage 5 Amplification Guide DNA Input ng Number of Cycles 50 5 20 6
7. molecular weight cell free DNA ChIP DNA 1 Single Indexing Reagents Index P B Notes before starting 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 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 until used 6 Indexing Reagents Indexing Reagents can be frozen and thawed no more than four times e MicroPlex Library Preparation kit v2 x48 48 indices is provided with a Single Index Plate SIP containing 48 ILLumina compatible single indexes each with a unique 8 nucleotide Sanger index
8. ol and allow to dry Prepare Library Amplification Master Mix as described in the table below Mix thoroughly with a pipette Keep on ice until used Library Amplification Master Mix Component Cap Color Volume Reaction Library Amplification Buffer Green 25 0 pL Library Amplification Enzyme Green 1 0 uL eer Free Water plus fluorescent Clear 4 0 pL Fluorescence dyes for detection and optical calibration are added when monitoring amplification in real time during cycling Please refer to the Real Time PCR Instrument s user manual for calibration dye recommendations The volume of detection and calibration dyes plus nuclease free water should not exceed 4 uL If a regular thermal cycler is used there is no need to add the dyes use 4 uL of nuclease free water Example EvaGreen Fluorescein dye mix Prepare by mixing 9 1 v v ratio of EvaGreen Dye 20X in water Biotium CAT NO 31000 T and 1 500 diluted Fluorescein Calibration Dye Bio Rad Laboratories CAT NO 170 8780 add 2 5 pL of this mix and 1 5 uL of nuclease free water per reaction Remove the seal on the PCR plate or open the tubels Add 30 uL of Library Amplification Master Mix to each well or tube Add 5 uL of the appropriate Indexing Reagent to each well or tube Note For 48 rxns 48 indices kit follow the SIP handling instructions section B 7 of this quick protocol to avoid index cross contamination Mix t
9. rom the thermal cycler and centrifuge briefly 9 Continue to the Library Synthesis Step Il Library Synthesis Step 1 Prepare Library Synthesis Master Mix as described in the table below Mix thoroughly with a pipette Keep on ice until used Template Preparation Master Mix Component Cap Color Volume Reaction Library Synthesis Buffer Yellow Tub Library Synthesis Enzyme Yellow Tul 2 Remove the seal on the plate or open the tube s 3 Add 2 uL of the Library Synthesis Master Mix to each well or tube 4 Mix thoroughly with a pipette Note Final volume at this stage is 15 uL 5 Seal the PCR plate using proper sealing film or tightly cap the tube s 6 Centrifuge briefly to collect contents to the bottom of each well or tube 7 Return the plate or tube s to the thermal cycler with a heated lid set to 101 C 105 C Perform Library Synthesis Reaction using the conditions in the table below Library Synthesis Reaction Temperature Time 22 C 40 min 4 C Hold lt 30 min 8 Remove the plate or tube s from the thermal cycler and centrifuge briefly 9 Continue to the Library Amplification Step Ill Library Amplification Step 1 Remove the Indexing Reagents from the freezer and thaw for 10 min on bench top Prior to use centrifuge the Indexing Reagents to collect the contents at the bottom For 48S kit wipe SIP foil seal with 70 ethan
10. used wipe the seal with 70 8 Low level multiplexing Select ethanol and let it dry completely Replace the plastic lid and return the plate to its sleeve and store at 20 C index combinations that meet the Illumina recommended compatibility requirements For MicroPlex Library Preparation kit v2 x48 48 indices kit multiplexing less than the full set of 48 libraries is possible on MiSeq only because MiSeq RTA v1 17 28 and later can process low plexity index reads For more information on multiplexing and index pooling please refer to Appendix 1 of the MicroPlex Library Preparation kit v2 Instruction Manual at www diagenode com 9 Index Plate Maps 000000000080 2000008600086 8 0000000000080 1000008000006 80 EL l 1 1 i OO 1 1 5 TO1 1S 1 1 i 1 O1 1 1 OOS lO L 1 C1 1 1 Cle 1S 1 1 1 Be TE Nay ai Ra 8 NO A Single Index Plate 48 rxns 48 indices Well Sequence Well Sequence Well Sequence Well Sequence Al ATCACGTT B1 GGTTGT Cl GCGATCT D1 GGCACAAC A2 CGATGTTT B2 CTCGGT C2 TCCTGCT D2 TCTCACGG A3 TTAGGCAT B3 AAGCGTI C3 AGTGACT D3 TCAGGAGG A4 TGACCACT B4 CCGTCT C4 ACAGGAT D4 TAAGTTCG A5 ACAGTGGT B5 GTACCT c5 CCTCAAT D5 TCCAGTCG Ab GCCAATGT B6 TCTGTG C GTGGTTG D TGTATGCG A7 CAGATCTG B7 CTGCTG C7 AGTCTTG D7 TCATTGAG A8 ACTTGATG B8 TGGAGGT C8 TCCATTG D8 TGGCTCAG A9 GATCAGCG B9 CGAGCGT C9 CGAAG
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