ThruPLEX® Plasma-seq Kit Quick Protocol
Contents
1. The 12S Kit is provided with 12 Indexing Reagents pre dispensed in tubes They have sufficient reagents for up to 8 uses and contain 8 nucleotide Sanger indexes that share the same sequences in the first 6 bases as the Illumina TruSeq LT indexes ADOO1 through ADO12 The 48S Kit is provided with a Single Index Plate SIP containing 48 Ilumina compatible single indexes each with a unique 8 nucleotide Sanger index sequence Each well has sufficient volume for a single use The 96D Kit is provided with a Dual Index Plate DIP containing 96 IIlumina compatible dual indexes Each well has sufficient volume for a single use and contains a unique combination of Illumina s 8 nucleotide TruSeq HT i5 and i7 index sequences 7 Index Plate Handling Instructions The Index Plate is sealed with pierceable sealing foil Follow the instruction below to avoid cross contamination Thaw the Index Plate 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 M Pierce the seal above each well containing the specific index combination with a clean 20 uL pipette tip with filter barrier discard the tip M Use a new pipette 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 f2. 9 199 8 728 737 and corresponding foreign patents Additional patents are pending TVA QAM 186 001 RUBICON GENOMICS B Quick Protocol I Template Preparation Step 6 Centrifuge briefly to collect contents to the bottom of each well Mix thoroughly with a pipette Avoid introducing excessive air 1 Add 10 uL of DNA sample to each well of a PCR plate or tube or tube bubbles If desired include control samples 7 Return the plate or tube s to the thermal cycler with a heated Note Final volume at this stage is 50 uL 2 Depending on the number of reactions prepare the Template lid set to 101 C 105 C Perform Library Synthesis Seal the plate or tube s tightly and centrifuge briefly to collect Preparation Master Mix as described in the table below Mix Reaction using the conditions in the table below contents to the bottom of each well or tube thoroughly with a pipette Keep on ice until used Library Synthesis Reaction Return plate or tube s to the real time PCR thermal Template Preparation Master Mix cycler thermal cycler with a heated lid set to 101 C 105 C Cap Color Volume Rxn Perform Library Amplification Reaction using the cycling Template Preparation Buffer Red 4u Hold lt 30 min conditions from the tables below Template Preparation Enzyme Red TL 8 Remove the plate or tube s from the thermal cycler and E Caution Ensure that the thermal cycler does not have a 3 To each 10 uL sample from step 1 abov
3. Aamp Qiagen GMBH QAM 186 001 www rubicongenomics com resources manuals F ATN RUBICON GENOMICS
4. ThruPLEX Plasma seq Kit Quick Protocol ThruPLEX Plasma seq is built with ThruPLEX chemistry to generate high quality DNA libraries from cell free DNA extracted from plasma samples Each kit contains all necessary reagents for preparing indexed Illumina NGS libraries including optimized Illumina compatible adapters and indexing reagents Each kit provides sufficient reagents for manual use up to 4 separate times For more information please visit www rubicongenomics com products ThruPLEX Plasma seq For detailed protocol refer to the ThruPLEX Plasma seq Kit Instruction Manual at www rubicongenomics com resources manuals Storage Store kit at 20 C upon arrival Technical support Call 734 677 4845 9AM 5 30PM Eastern Time or contact support rubicongenomics com Kit Contents Input DNA Sample Requirements Cap 125 Ki 485 Ki DRE J J Requirement EEN ea NO R400490 ak ta NO R400491 CAT NO R400492 Plasmas Template Preparation Buffer ulgpanetion Buffer 1 IT ue 1 1 Tube QTubes QTubes Cellfree DNA brary Synthesis Buffer Input Amount Input volume Input volume 10 pL 10 pL lt 0 1 mi EDTA EEEE RE indexing Reagents __ 12 Tubes 1 Single Index Plate 1 Duel ndex Plate equivalent methods A Notes Before Starting 1 Input DNA Sample Requirements See table above right QIAamp Circulating Nucleic Acid Kit Qiagen CAT NO 55114 is the recommended method for extracting cell free DNA fr
5. e add 5 uL of the centrifuge briefly denaturing step programmed until Stage 3 Template Preparation Master Mix 9 Continue to the Library Amplification Step Library Amplification Reaction 4 Mix thoroughly with a pipette Ill Library Amplification Step Stage Temperature Time No of Cycles Note Final volume at this stage will be 15 uL 1 Remove the Indexing Reagent from the freezer and thaw for 10 Extension amp _ l Cleavage 5 Seal the PCR plate using proper sealing film or tightly cap the min on bench top Prior to use centrifuge the Indexing Reagents tube s to collect the contents at the bottom Wipe the Index Plate foil ee 98 C IA a s 6 Centrifuge briefly to collect contents to the bottom of each well seal with 70 ethanol and allow to dry Addition of 4 67 C 20s 4 or tube 2 Prepare Library Amplification Master Mix as described in Indexes 72 C 40s 7 Place the plate or tube s in a thermal cycler with a heated lid set oa below Mix thoroughly with a pipette Keep on ice library 98 C 20s 5 tol to 101 C 105 C Perform the Template Preparation oe Amplification 2 72 C 50s plies Reaction using the conditions in the table below Library Amplification Master Mix i Cap Color_ Volume itxn aT SE ET ee Template Preparation Reaction eee 21 5 pL cquire fluorescence data at this step if monitoring in real time Library Amplification Enzyme 1 0 uL E Selecting the optimal number of amplification cycles Fluorescence Dyes P
6. ing with Qubit quantified cell free DNA and following this Library Synthesis Buffer 2 5 UL of this mix per reaction protocol the typical yields range from 500 ng to 1000 ng Library Synthesis Enzyme 2 5 uL 3 Remove the seal on the PCR plate or open the tube s Remove the plate or tube s from the thermal cycler and 2 Remove the seal on the plate or open the tube s 4 Add 25 uL of Library Amplification Master Mix to each centrifuge briefly 3 Add 5 uL of the Library Synthesis Master Mix to each well well or tube M Note At this stage samples can be processed for next or tube 5 Add 5 uL of the appropriate Indexing Reagent to each well or generation sequencing NGS immediately or stored frozen at 4 Mix thoroughly with a pipette tube 20 C for up to 2 weeks For instructions and recommendations Note Final volume at this stage is 20 uL E Note For the 48S and 96D Kits follow the Index Plate handling on library pooling purification quantification and sequencing 5 Seal the PCR plate using proper sealing film or tightly cap the instructions section A 7 to avoid index cross contamination please refer to the ThruPLEX Plasma seq Kit Instruction Manual at tube s Technical Support Call 734 677 4845 9AM 5 30PM EST or contact support rubicongenomics com Trademarks ThruPLEX Illumina TruSeq Illumina Inc EvaGreen Biotium Inc Agencourt AMPure Beckman Coulter Inc Qubit Life Technologies Corporation Ql
7. o 2 5 uL The number of PCR cycles required at Stage 5 of the Library or Nuclease Free Water Amplification Reaction is dependent upon the amount of input E Fluorescence Dyes for detection and optical calibration are DNA and the thermal cycler used The table below provides the 8 Remove the plate or tube s from the thermal cycler and added when monitoring amplification in real time during cycling suggested number of PCR cycles at Stage 5 for different input centrifuge briefly Refer to the real time PCR instrument s user manual for amounts of cell free DNA 9 Continue to the Library Synthesis Step calibration dye recommendations The volume of detection and Stage 5 Amplification Guide Il Library Synthesis Step calibration dyes should not exceed 2 5 uL If a regular thermal DNA Input ng Number of Cycles cycler is used there is no need to add the dyes use 2 5 uL of 30 5 1 Prepare Library Synthesis Master Mix as described in the nuclease free water noie below Mix thoroughly with a pipette Keep on ice until E Example EvaGreen Fluorescein dye mix Prepare by ee ee es si mixing 9 1 v v ratio of EvaGreen Dye 20X in water Biotium Yield The amount of amplified library can vary depending upon Library Synthesis Master Mix CAT NO 31000 T and 1 500 diluted Fluorescein Calibration sample condition composition and thermal cycler used When Cap Color Volume Rxn Dye Bio Rad Laboratories CAT NO 170 8780 add 2 5 uL start
8. om plasma 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 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 cycling process by using proper seal caps as evaporation may reduce reproducibility 4 Positive and Negative Controls If desired include a positive control DNA 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 Prepare 5 excess of each master mix to allow for pipetting losses Each kit contains sufficient reagents to prepare master mixes up to 4 separate times Keep all enzymes buffers and master mixes on ice until use M Transfer the enzymes to ice just prior to use and centrifuge briefly to collect contents at the bottom of the tube Thaw the buffers vortex briefly and centrifuge prior to use E The 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 M
9. rom 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 Wipe the seal with 70 ethanol and let it dry completely Replace the plastic lid return the plate to its sleeve and store at 20 C 8 Low Level Multiplexing Select appropriate index combinations that meet Illumina recommended compatibility requirements For more information on multiplexing and index pooling refer to the ThruPLEX Plasma seq Kit Instruction Manual at www rubicongenomics com resources manuals 9 Index Sequences and Index Plate Maps Refer to the ThruPLEX Plasma seq Index Guide at www rubicongenomics com resources manuals 10 Library Purification Quantification and Sequencing For instructions and recommendations refer to the ThruPLEX Plasma seq Instruction Manual at www rubicongenomics com resources manuals ThruPLEX Plasma 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 Plasma seq 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 Plasma seq is protected by U S Patents 7 803 550 8 071 312 8 39
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