Protocol Guide 49604 - Asuragen
Protocol Guide49604For Research Use Only.Not for use in diagnostic procedures.49602, 4960348 - 192Asuragen, Inc.2150 Woodward St., Ste. 100Austin, TX 78744-1840USA 1.512.681.520049604v1 DRAFT – NOT Effective
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolTable of ContentsQuantideX NGS RNA Lung Cancer Kit Protocol Overview . 3QuantideX NGS RNA Lung Cancer Kit Components . 4Kit Configuration . 5Required Materials Not Provided . 5Optional Equipment Not Provided . 6Intended Use. 6Fusion Targets . 6Test Principle . 7Limitations . 7Warnings and Precautions . 8QuantideX NGS RNA Lung Cancer Kit Library Prep Protocol . 9Overview/Notes . 9Reverse Transcription (RT) [Box 1] . 9RNA Assay [Box 1] . 10Gene-Specific PCR [Box 2]. 11Tagging PCR [Box 2 and Box 3] . 11Library Purification [Box 4] . 12Library Quantification [Box 5] . 13Pool, Denature, Dilute, Load [Box 6] . 15Generation of MiSeq Sample Sheet. 16Data Analysis . 16Expected Results of Kit Controls/Standards and QC Steps . 16Notice to Purchaser . 16Symbol Legend . 17Reference . 17Appendix 1: Index Codes . 18Appendix 2: Targeted Regions . 20Appendix 3: Other Targets . 23Related Protocols PC-0249, QuantideX NGS Reporter User Guide, available at http://quantidex.asuragen.comMiSeq System User Guide, available at http://support.illumina.com/Use these protocols in conjunction with the following protocol to successfully execute the QuantideX NGS RNA Lung Cancer Kit library prep workflow.Contact Asuragen Technical Support if any problems are encountered when using this product:support@asuragen.com and at 1.512.682.520049604v1 – 20Oct16Page 2 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolQuantideX NGS RNA Lung Cancer Kit Protocol Overview Kit: QuantideX NGS RNA Assay (Lung) [Box 1]ReverseTranscription(RT)Temp42 C93 C4 C 4 µL QuantideX RT Buffer 1 µL QuantideX RT Enzyme Mix 5 µL RNA/TNA sample, NTC or RNA Positive Fusion Ctrl (provided)Time45 min10 minhold Kit: QuantideX NGS RNA Assay (Lung) [Box 1]RNA Assay 5 µL 2X qPCR Master Mix 2.5 µL Primer/Probe Mix (Lung) 0.5 µL 20X ROX (if needed) 2 µL RT product or qPCR Standards 1 - 4 (Lung)Temp95 C95 C60 CTemp95 C95 C60 C95 C72 C72 C4 C Kit: QuantideX NGS RNA Lung Panel [Box 2] 5 µL 2X Amplification Master MixGene-SpecificPCR 1 µL RNA Lung Primer Mix 4 µL RT product (Suggested input 400 functional copies) Kits: QuantideX NGS RNA Lung Panel [Box 2] &QuantideX Codes - Set A/B/C/D [Box 3]Tagging PCR 7.5 µL 2X Index Master Mix 5.5 µL Index Code (AIL001-AIL192) 2 µL Gene-Specific PCR productTimeCycling5 min5 sec40 cycles60 secqPCR using FAM channelTemp95 C95 C55 C72 C72 C4 CTime5 min15 sec4 min15 sec4 min10 minholdTime5 min30 sec30 sec60 sec10 minholdCycling2 cycles23 cycles-Cycling10 cycles- Kit: QuantideX NGS Library Pure Prep [Box 4]LibraryPurification 11 µL Library Pure Prep Beads10 µL Tagging PCR product2 X 100 µL washes w/ EtOH-diluted Wash BufferElute in 20 µL Elution Buffer Kit: QuantideX NGS Library Quant [Box 5] 5 µL 2X qPCR Master Mix 2.5 µL LQ Primer/Probe MixMix, pellet beads on magnet, removesupernatantLeave plate on magnetElute 5 min, recover pure productsTempTimeCycling95 C5 min95 C5 sec35 cycles60 C60 secqPCR using FAM channelLibrary 0.5 µL 20X ROX (if needed)Quantification 2 µL Diluted purified product (diluted 10,000-fold in LQ Diluent) or LQ Standards 1 - 4 Kits: QuantideX NGS Sequencing Reagents [Box 6] & Illumina MiSeq Reagent KitPool,Denature,Dilute, Load49604v1 – 20Oct16 15 µL 2.5 nM normalized library pool from stock purified products 3 µL 1 nM PhiX Control V3, 2 µL 1 N NaOH; denature for 5 min at room temp, snap cool on ice Dilute 8 µL of the denatured library into 992 µL pre-chilled HT1 buffer Dilute 4 µL Read 1, Index Read, & Read 2 Primers in 636 µL HT1 buffer individually, load MiSeq Page 3 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolQuantideX NGS RNA Lung Cancer Kit ComponentsReference No.49589DescriptionCap color, ifapplicableQuantideX NGS RNA Assay (Lung) [Box 1]Vol/RxnssupportedStorage Temperature48 Reactions-15 to -30 C145435QuantideX RT Enzyme Mixgreen48 µL-15 to -30 C145436QuantideX RT Buffergreen192 µL-15 to -30 C145442RNA Fusion Positive Ctrl (Lung)clear240 µL-15 to -30 CqPCR Standards 1 - 4 (Lung)white32 µL each-15 to -30 C1454442X qPCR Master Mixyellow360 µL x 2-15 to -30 C145443Primer/Probe Mix (Lung)blue280 µL-15 to -30 C14544120X ROXamber56 µL2 to 8 C (after 1st use)48 Reactions-15 to -30 C145437-4049588QuantideX NGS RNA Lung Panel [Box 2]1453482X Amplification Master Mixgreen240 µL150016RNA Lung Primer Mixgreen48 µL1453612X Index Master Mixyellow360 µL49553-49556QuantideX NGS Codes - Set A/B/C/D*[Box 3]150004-150007 Index Codes (ILM) - Set A/B/C/D49551QuantideX NGS Library Pure Prep [Box 4]145351Library Pure Prep Beads145352Wash Buffer145353Elution Buffer49590rack of codesamberbottleblueQuantideX NGS Library Quant [Box 5]192 Reactions-15 to -30 C22 µL2 to 8 C (after 1st use)48 Reactions2 to 8 C530 µL2.5 mL2 to 8 C1200 µL x 248 Reactions-15 to -30 C19.2 mL2 to 8 C (after 1st use)145445LQ Diluent1454442X qPCR Master Mixyellow360 µL x 2-15 to -30 C145447LQ Primer/Probe Mixviolet280 µL-15 to -30 C14544120X ROXamber56 µL2 to 8 C (after 1st use)LQ Standards 1 - 4blue32 µL-15 to -30 C8 Runs2 to 8 C145449-5249557bottle-15 to -30 CQuantideX NGS Sequencing Reagents[Box 6]145365Sequencing Diluentblue1200 µL150001Read 1 Sequencing Primersgreen32 µL150002Index Read Sequencing Primersgreen32 µL150003Read 2 Sequencing Primersgreen32 µL2 to 8 C*Only one of the four sets of Index Codes are included when ordering the QuantideX NGS RNA Lung Cancer Kit.See Appendix 1 for more details regarding the QuantideX NGS Codes.Notes: Box numbers are included on box labels. Reactions supported assumes approximately using 15% overagewhen preparing reaction master mixes.The QuantideX NGS Reporter (49562), a bioinformatics solution for analyzing MiSeq output files, is included inthe purchase of the kit. See the QuantideX NGS Reporter User Guide (PC-0249), available athttp://quantidex.asuragen.com/ for download.49604v1 – 20Oct16Page 4 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolKit ConfigurationRequired Materials Not ProvidedSuggested part numbers have been provided for reagents and consumables, but equivalent products may be used.Consult the MiSeq System User Guide for additional materials required to use Illumina MiSeq instrument.o Nuclease-free watero 96-100% Ethanolo 1 N Sodium Hydroxideo PhiX Control v3 (Illumina 15017666)o Round-bottom plates (Evergreen 290-8117-01R)o Thermal cyclero PCR plates and sealso qPCR instrument†o Optical plates and sealso Magnetic plate stand (Ambion AM10027)o Micronic tube decapper (Univo SR008, or equiv.)o Illumina MiSeq Desktop Sequencer and MiSeq Reagent KitOptimal number of samples for each MiSeq Reagent KitReagent KitCatalog#Number of Reads ‡MiSeq Reagent Kit v3,600 cyclesMiSeq Reagent Kit v2,500 cyclesMS-102-300325 millionRecommended Number ofSamples with Kit*8-48MS-102-200315 million4-24† If an ABI 7500 (Fast or Standard) or Roche 480 qPCR instrument is unavailable for qPCR, consult Asuragen Tech Support forguidance on use of other platforms.‡ This information is provided by Illumina. For more information, consult Illumina for MiSeq System Specification.* The number of samples for each MiSeq reagent kit is recommended to achieve the optimized QuantideX NGS RNA Lungdata output. Consult Asuragen Tech Support for further assistance.49604v1 – 20Oct16Page 5 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolOptional Equipment Not ProvidedoFlatbed or handheld scanner with Data Matrix 2D barcode reading capabilitiesIntended UseThe QuantideX NGS RNA Lung Cancer Kit is intended for the detection of clinically-relevant content for RNAtargets common to lung cancer including both fusions as well as mRNA expression profiling for relevant genesfrom RNA or TNA purified from human tissue, FFPE or cell-lines. The kit covers 107 specific RNA fusions (shownbelow), MET exon 14 skipping, 3’-5’ imbalance ratios for 5 RNA expression markers (See Appendix 3) involved inoncogenic translocations, and 23 expression markers that are semi-quantitatively evaluated against a set of lowvariation reference genes. The kit supports multiplex next-generation sequencing analysis with an Illumina MiSeq instrument. The kit includes software (QuantideX NGS Reporter, 49562) that analyzes MiSeq data filesfor the identification of fusion targets using a locally integrated bioinformatic pipeline and companion datavisualization tools.Fusion Targets3' GenePartner5' GenePartnerCOSMIC ID3' GenePartner5' GenePartnerCOSMIC ID3' GenePartner5' GenePartnerCOSMIC 64MBIPAXLROS1EZR49604v1 – 20Oct16COSF1267Page 6 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit Protocol3' GenePartner5' GenePartnerCOSMIC ID3' GenePartner5' GenePartnerCOSMIC ID3' GenePartner5' GenePartnerCOSMIC SF1357ROS1SDC4ROS1TPM3COSF1265Note: Shading is intended to assist with visual grouping of the common 3’ Gene Partners.Test PrincipleThe kit includes reagents for RT, cDNA QC, targeted enrichment, index codes, library purification, quantification,and an easy-to-use bioinformatics software solution. Both wet and dry bench processes are integrated within asimple workflow optimized for use with low-quality and low-quantity RNA/TNA samples isolated from FFPE(Formalin-Fixed, Paraffin-Embedded), FNA (Fine Needle Aspiration) tumor biopsies, fresh frozen (FF) tissue, andcell lines.Each kit component serves an essential role in the library prep workflow: QuantideX NGS RNA Assay (Lung) – Reagents for functional RNA RT, subsequent quantification, and sampleQC assessment. Additionally, the box contains the RNA Fusion Positive Ctrl (Lung).QuantideX NGS RNA Lung Panel – PCR primers and reagents that support single-well multiplex PCRenrichment across all targets.QuantideX NGS Codes: Set A/B/C/D – Dual Index code oligonucleotide mixtures specific for the MiSeq. Thesedual-index code primer mixes are formulated in racks of 48 codes.QuantideX NGS Library Pure Prep – A proprietary magnetic bead chemistry that provides size selection andpurification of the amplified libraries.QuantideX NGS Library Quant – An assay that enables accurate assessment of purified libraries using aquantitative real-time PCR method.QuantideX NGS Sequencing Reagents – Custom sequencing primer mixtures for QuantideX NGS and standardIllumina library analysis.QuantideX NGS Reporter –An easy to use, integrated informatics processing and data reporting pipelinebased on proprietary alignment and variant scoring algorithms.LimitationsSample Input The concentration of the cDNA sample must be determined as functional or amplifiable copies permicroliter (cp/µL).o Use the QuantideX NGS RNA Assay to obtain copy number concentrations for each sample’s RTproduct.o Samples with 100 cp/µL may be analyzed, but are at-risk for inaccuracies due to the reducedtemplate diversity available within that sample for amplification.o For best results, load 100 cp/µL (Total: 400 amplifiable copies) but 6000 cp/µl into the GeneSpecific PCR enrichment.49604v1 – 20Oct16Page 7 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolRare Single Nucleotide Polymorphisms (SNPs) The QuantideX NGS library preparation method is based on PCR amplification of targeted regions ofcancer-associated genes (see Appendix). The presence of rare SNPs in the primer binding region mayresult in reduced amplicon yield or allele dropout.Thermal cycler and qPCR Instrument platforms The Kit has been verified for use on the ABI 9700 and Veriti instruments with default ramp rates. Use ofother instrument platforms other than those mentioned here is NOT supported.1The RNA Assay and LQ qPCR protocol has been verified for use on the ABI 7500, ABI 7500 Fast in standardmode and Roche LC480 & Cobas Z480 instruments. Analysis in fast mode or use of other instrumentplatforms other than those mentioned here is NOT supported.2Reagent Stability The reagents should be used within their labeled expiry and stored according to kit component table.Reagents stored frozen (-15 to -30 C) are formulated to support eight (8) freeze-thaw cycles.MiSeq Instrument Control The PhiX sequencing control is highly recommended for inclusion in each NGS run for clusterdiversification and to aid in troubleshooting.Instrument/Platform Compatibility This protocol was written to support MiSeq instrument analysis. Specific modifications may be requiredfor other Illumina Sequencing platforms (e.g. NextSeq system).A separate protocol guide may be used to describe use of these reagents for alternative platforms.Warnings and Precautions Use proper PPE. Wear appropriate protective eyeglasses, protective gloves, and protective clothing whenworking with these materials.Follow Universal Precautions in compliance with OSHA 1910:1030, CLSI M29, or other applicable guidancewhen handling human samples.Use nuclease-free filter pipette tips and nuclease-free tubes.Seal plates in a safe and appropriate manner to reduce likelihood of evaporation or cross contaminationof wells during library preparation. Automated heat sealing using peelable foil seals is recommended.o The BioRad PX1 Heat Sealer (185 C, 3 seconds) with Eppendorf Twin-Tec 96 well plates andpeelable foil heat seals (BioRad Ref #1814045), or equivalent, has been verified for efficacy.PCR carry-over contamination can result in false-positive signals. Use appropriate precautions in samplehandling, workflow, and pipetting.oSeparation of template (i.e. sample handling) and non-template (i.e. master mix formulation)handling is highly recommended to reduce the probability of introducing contamination into theworkflow.Do not combine components from different reagent lots.Do not let beads dry out during library Purification to mitigate risk of sample loss/low library yield.oTo reduce drying effects, divide the plate into sets of 2 or 3 columns at a time when purifyingfor large sample batches.Prior to use, ensure that all instruments are calibrated according to the manufacturer’s instructions.When working with less than 8 samples, it is recommended to repeat Library Quant afterpooling/normalizing libraries prior to loading the MiSeq.1If interested in using a different library quantitation (qPCR) platform, please contact Asuragen Tech Support forguidance.49604v1 – 20Oct16Page 8 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolQuantideX NGS RNA Lung Cancer Kit Library Prep ProtocolOverview/Notes Track sample ID, amplifiable copy number, and assigned index code carefully throughout the procedure.All three are critical for successful sequencing analysis.Use this protocol in conjunction with Illumina’s MiSeq System User Guide.All master mixes and reactions may be prepared at room temperature.When combining reagents as instructed, pipette up and down 3-5 times with each addition to ensure afull dispense.Replicate testing of samples in qPCR assays is not required or recommended.Library prep can be safely stopped after each major reaction in the procedure.o After the reaction is complete, store sample plate(s) at 2 to 8 C for short term ( 24 hrs) or longterm at -15 to -30 C. Keep all reaction plates until sequencing data has been obtained.o When ready to proceed, bring plate to room temperature with other required reagents.Prior to each reaction,o Bring each required reagent and/or plate to room temperature for at least 15 minutes.o Briefly centrifuge each plate before use.o Briefly vortex and centrifuge each component prior to opening.Exception: Do not centrifuge the Library Pure Prep Beads.ReverseTranscriptionRNA AssayGene-SpecificPCRTagging PCRLibraryPurificationLibraryQuantificationPool, Denature,Dilute, LoadReverse Transcription (RT) [Box 1]Instrument run time: 55 min1. Prepare an RT reaction master mix in a clean microcentrifuge tube.A. Add the reagents to the tube in the order listed;DescriptionVol. (µL)volumes are shown per reaction.QuantideX RT Buffer4B. Prepare sufficient master mix for the totalQuantideX RT Enzyme Mix1number of samples tested, including anyTotal master mix per well5necessary controls (NTC and RNA FusionPositive Ctrl).C. Mix the master mix with gentle flicking/vortexing, briefly centrifuge to collect contents.2. Aliquot 5 µL of RT master mix to separate wells in a 96-well reaction plate.Including the3. Add 5 µL of each RNA/TNA sample of interest to separate wells containing RTsuggested controlsmaster mix, mix by pipetting.in each library prepbatch helps to4. Add 5 µL of each control to separate wells containing RT master mix, mix byensure run validity,pipetting.monitor operatorA.RNA Fusion Positive Ctrlproficiency, andtrack performanceB. NTC (nuclease-free water)trends over time.5. Seal the plate and briefly centrifuge to collect contents.Do not vortex plate to reduce risk of contamination.6. Perform the 10 µL Reverse Transcription (RT) reaction on a thermal cycler with the following conditions:Temp.Time42 C45 min93 C10 min4 Chold49604v1 – 20Oct16Page 9 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolReverseTranscriptionRNA AssayGene-SpecificPCRTagging PCRLibraryPurificationLibraryQuantificationPool, Denature,Dilute, LoadRNA Assay [Box 1]Instrument run time: 1.5 hours1. Prepare a qPCR master mix in a clean microcentrifuge tube.DescriptionVol. (µL)A. Add the reagents to the tube in the order listed;2X qPCR Master Mix5.0volumes are shown per reaction.Primer/Probe Mix2.5B. Prepare sufficient master mix for the total number of20X ROX†0.5RT products, plus the 4 qPCR standards in duplicate (#Total master mix per well8.0of RT products 8).† If using a qPCR instrument that does notC. Mix the master mix with gentle flicking/vortexing,utilize a passive reference, substitute thisthen pulse spin to collect contents.volume with nuclease-free water.D. After first use, store 20X ROX at 2 to 8 C2. Aliquot 8 µL of qPCR master mix to separate wells in a 96-well optical PCR plate.Example of a 63. Add 2 µL of each qPCR Standard to separate wells in duplicate (8 wells total)reaction plate layout:123and mix by pipetting.qPCRASample14. Add 2 µL of each RT product to separate wells and mix by pipetting.Std. 1qPCR5. Seal plate with optical seal, then centrifuge plate to collect contents.BSample2Std. 1qPCRDo not vortex plate to reduce risk of contamination.CSample3Std. 26. Perform the 10 µL qPCR reaction on a qPCR instrument with parameters below:--Collect data during 60 C stepTemp.TimeABI 7500 instruments95 C5 mino Standard mode95 C5 seco FAM detector, no quencher,60 C60 seco ROX Passive Reference;o Auto Baseline;o Manual Threshold: 0.3Roche LC480 instruments:o FAM detectoro Fit Points analysiso Suggested analysis setting (verify and adjust per run):Background 3-9,Noise band STD Multiplier 36,Threshold Auto;Cycling40cyclesDEFGHqPCRSample4Std. 2qPCR RNA FusionStd. 3 Pos. Ctrl.qPCRNTCStd. 3qPCRStd 4qPCRStd 47. AnalysisA. Determine Copy InputPlot a standard curve of the qPCR Standards, using log (concentration) as the x-axis, and FAM Cqvalues for each standard as the y-axis.Using the equation of the best-fit linear line, calculate concentration (cp/µL) for each sample.Record all sample copy numbers for analysis by the QuantideX Reporter.B. Input recommendations into QuantideX NGS RNA LungDescriptionConc. (cp/µL)library prep:qPCR Standard 1 (Lung)6250qPCR Standard 2 (Lung)1250 100 cp/µL: may proceed to library prep, but at-risk forqPCRStandard3(Lung)250call inaccuracies due to potentially limiting functionalqPCR Standard 4 (Lung)50template quantities 100 cp/µL: recommended input for library prep 6000 cp/µL: dilute an aliquot of the sample 10-fold in nuclease-free water for use in libraryPrep (e.g. 2 µL sample in 18 µL water).49604v1 – 20Oct16Page 10 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolReverseTranscriptionRNA AssayGene-SpecificPCRTagging PCRLibraryPurificationLibraryQuantificationPool, Denature,Dilute, LoadGene-Specific PCR [Box 2]Instrument run time: 2.5 hours1. Prepare a GS PCR master mix in a clean microcentrifuge tube.A. Add the reagents to the tube in the order listed;DescriptionVol. (µL)volumes are shown per reaction.2X Amplification Master Mix5RNA Lung Primer Mix1B. Create enough master mix for all RT products (samplesof interest plus any controls included).Total master mix per well6C. Gently vortex to mix, and briefly centrifuge the GS PCRmaster mix.2. Aliquot 6 µL of the GS PCR master mix into separate wells of a clean 96-well plate.3. Add 4 µL of each RT product to separate wells containing GS PCR master mix, mix by pipetting.Note: Recommended cDNA input range is 400 copies ( 100 cp/µL)4. Seal the plate, briefly centrifuge the plate to collect contents.Temp.TimeCyclingAutomated heat sealing using peelable foil seals is recommended.95 C5 minDo not vortex plate to reduce risk of contamination.95 C15 sec2 cycles5. Perform the 10 µL GS PCR reaction on a thermal cycler with the indicated60 C4 mincycling conditions.95 C15 sec23 cycles72 C4 min72 C10 min4 Chold-ReverseTranscriptionRNA AssayGene-SpecificPCRTagging PCRLibraryPurificationLibraryQuantificationPool, Denature,Dilute, LoadTagging PCR [Box 2 and Box 3]Instrument run time: 45 min1. PreparationA. Centrifuge the rack of index codes for 1 minute at 2000 x g prior to each use.B. If needed, scan rack of codes with a barcode scanner to ensure correct positions of index codes.Refer to the provided COA as needed.C. Assign each sample/control from GS PCR a unique Index Code (AIL001-AIL192), and record thisinformation by plate location and sample ID.Critical: Each sample must be assigned (and receive) a different index code.2. In a new 96-well plate, for each GS PCR product:A. Aliquot 7.5 µL 2X Index Master MixB. Add 5.5 µL Index Code (AIL###, unique to each well)3. Following brief centrifugation, transfer 2 µL GS PCR product to corresponding wells containing IndexCodes and Master Mix. Mix by pipetting.49604v1 – 20Oct16Page 11 of 24
Asuragen QuantideX NGS RNA Lung Cancer Kit ProtocolImportant: Ensure the correct Index Code was added to the appropriate well according to thepreviously-made assignments and that each well received a different Index Code.4. Seal the plate, and briefly centrifuge to collect contents.Automated heat sealing using peelable foil seals is recommended.Do not vortex plate to reduce risk of contamination.5. Perform the 15 µL Tagging PCR reaction on a thermal cycler with the indicated cycling conditions.Temp.95 C95 C55 C72 C72 C4 CReverseTranscriptionRNA AssayGene-SpecificPCRTagging PCRLibraryPurificationTime5 min30 sec30 sec60 sec10 minholdLibraryQuantificationCycling10cycles-Pool, Denature,Dilute, LoadLibrary Purification [Box 4]1. Initial SetupA. If this is the first use of the kit, add 10 mL of 100% Ethanol to Wash Buffer bottle, cap the bottle,and mix well by inverting the bottle several times.10 mL of 100% ethanol must be added to the Wash Buffer bottle.Failure to add ethanol to the Wash Buffer will result in sample and reagent loss.B. Bring Library Pure Prep Beads to room temperature prior to useC. Fully re-suspend Library Pure Prep Beads with gentle vortexing for 30 - –5 seconds.DO NOT centrifuge the Library Pure Prep Beads.D. Ensure reagents and consumables are readily available before starting the purification. It isimportant to not let the beads dry out until the very end of the procedure.To reduce drying effects, divide the plate into sets of 2 or 3 columns at a time when doing thepurification for large sample batches.2. Bind the Tag PCR product to the magnetic beadsA. Aliquot 11 µL Library Pure Beads to each well in a clear, round-bottom, 96-well plate (EvergreenScientific: 290-8117-01R). Prime pipette tip to ensure full volume dispense.Note: To keep bead concentration consistent across the plate while aliquotting, intermittently capthe Library Pure Prep Beads after every 4 wells and gently pulse vortex.B. After brief centrifugation, add 10 µL Tag PCR products to the wells of beads, mix by pipetting untilmixture appears homogenous. 5 µL Tag PCR product will remain in Tag PCR plate.Note: Avoid creating bubbles while mixing.Note: Recommended use of multichannel for ease of work flow from this point onwards.3. Wash the bead-bound libraries:A. Place plate on magnetic stand (Ambion AM10027) to pellet beads out of solution ( 15 seconds).B. Leaving the plate on the magnetic stand, slowly remove and discard the clear supernatant ( 20µL) from each well, taking care to avoid the bea
Consult the MiSeq System User Guide for additional materials required to use Illumina MiSeq instrument. o Nuclease-free water o 96-100% Ethanol o 1 N Sodium Hydroxide o PhiX Control v3 (Illumina 15017666) o Round-bottom plates (Evergreen 290-8117-01R) o Thermal cycler o PCR plate
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