SureDirect Blood PCR Kit Manual

SureDirect Blood PCR KitCatalog #600920 (100 Reactions) and Catalog #600930 (500 Reactions)Agilent TechnologiesProtocolVersion B.0, April 2014WarrantyThe material contained in this document is provided “as is,” and is subject to being changed, with-out notice, in future editions. Fur-ther, to the maximum extent permitted by applicable law, Agi-lent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchant-ability and fitness for a particular purpose. Agilent shall not be lia-ble for errors or for incidental or consequential damages in con-nection with the furnishing, use, or performance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this doc-Safety NoticesCAUTIONA CAUTION notice denotes a haz-ard. 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Not for use in diagnostic procedures.In this Guide...123SureDirect Blood PCR KitThis guide describes an optimized protocol for using the SureDirect Blood PCR Kit to amplify targets of interest from blood or blood-derivative samples.Before You BeginThis chapter contains information (such as procedural notes, safety information, required reagents and equipment) that you should read and understand before you start an experiment.Analysis of Liquid Blood SamplesThis chapter describes the protocol steps for PCR analysis of targets of interest directly from liquid blood samples.Analysis of Dried Blood SamplesThis chapter describes the protocol steps for PCR analysis of targets of interest directly from blood samples dried on a solid support.3What’s New in Version4 B.0• Updated instructions for use of frozen blood samples (page 12)SureDirect Blood PCR KitContent1 Before You BeginProcedural Notes 8Safety Notes 8Reagents Required for the Protocol 9Contents of the SureDirect Blood PCR Kit 9Required Equipment for Analysis of Liquid Blood Samples 10Required Equipment for Analysis of Dried Blood Samples 102 Analysis of Liquid Blood SamplesPCR primer considerations 12Blood sample properties and sample quantity considerations 12Optimization parameters 13Step 1. PCR-amplify the target DNA from liquid blood samples 15Step 2. Recover the amplicons from lysed cell debris 19Step 3. Analyze the amplicons by gel electrophoresis 213 Analysis of Dried Blood SamplesPCR primer considerations 24Preparation and storage of dried blood samples 24Optimization parameters 25Step 1. PCR-amplify the target DNA from dried blood samples 26Step 2. Analyze the amplicons by gel electrophoresis 30SureDirect Blood PCR Kit 56 SureDirect Blood PCR KitSureDirect Blood PCR Kit Protocol1Before You BeginProcedural Notes 8Safety Notes 8Reagents Required for the Protocol 9Contents of the SureDirect Blood PCR Kit 9Required Equipment for Analysis of Dried Blood Samples 10Make sure you read and understand the information in this chapter and have the necessary equipment and reagents listed before you start an experiment.7Agilent Technologies1 Before You Begin 8Procedural NotesProcedural Notes• Follow your institution’s guidelines for safety procedures and other practices required for working with blood samples.• In general, follow Biosafety Level 1 (BL1) safety rules.• Ensure that reagent mixtures are thoroughly mixed, by pipetting up-and-down or by gentle vortexing, before distributing to the samples.Safety Notes• Wear appropriate personal protective equipment (PPE) when working in the laboratory.CAUTIONSureDirect Blood PCR KitBefore You Begin 1 RCReagent FormatSureDirect Blood PCSureDirect Blood PCR 2× MM (Master Mix provided at 2× concentration)tube with red capDMSO tube with green cap10× Control Primers + Template tube with clear capTable 2 SureDirect Blood PCR Kit ContentsReagents Required for the Protocoleagents Required for the Protocol1ontents of the SureDirect Blood PCR KitTable 1 Required Reagents for the SureDirect Blood PCR Kit protocolDescription Vendor and part numberSureDirect Blood PCR Kit* 100 Reactions 500 Reactions* See Table 2, below, for a list of included reagents.Agilent p/n 600920 p/n 600930Nuclease-free Water (PCR-grade) Cromasolv water, Sigma, p/n 27073, or equivalentPCR Primer Pair, desalted IDT, or equivalentR Kit 91 Before You Begin Required EquipRRIce bucket General laboratory supplierVortex mixer General laboratory supplier10equired Equipment for Analysis of Dried Blood SamplesTable 4 Equipment required for analysis of dried blood samplesDescription Vendor and part numberWhatman 903 Protein Saver Cards GE Healthcare, p/n 10534320Sample disc puncher with 2-mm ID bore GE Healthcare, p/n WB100007 or equivalentSterile forceps General laboratory supplierThermal Cycler Agilent SureCycler 8800, p/n G8800A or equivalent8-well PCR strip tubes Agilent, p/n 410092Tube cap strips (8 domed caps per strip) Agilent, p/n 410096P20 and P200 pipettes Pipetman or equivalentVortex mixer General laboratory supplierment for Analysis of Liquid Blood Samplesequired Equipment for Analysis of Liquid Blood SamplesTable 3 Equipment required for analysis of liquid blood samplesDescription Vendor and part numberThermal Cycler Agilent SureCycler 8800, p/n G8800Aor equivalent8-well PCR strip tubes Agilent, p/n 410092Tube cap strips (8 domed caps per strip) Agilent, p/n 410096Centrifuge with strip tube-compatible rotor, such as Eppendorf 5424 benchtop microcentrifuge with strip-tube rotor VWR p/n 93000-196 (centrifuge) and p/n 80094-150 (rotor), or equivalentP20 and P200 pipettes Pipetman or equivalentIce bucket General laboratory supplierSureDirect Blood PCR KitMicrocentrifuge General laboratory supplierSureDirect Blood PCR Kit Protocol2Analysis of Liquid Blood SamplesPCR primer considerations 12Blood sample properties and sample quantity considerations 12Optimization parameters 13Step 1. PCR-amplify the target DNA from liquid blood samples 15Step 2. Recover the amplicons from lysed cell debris 19Step 3. Analyze the amplicons by gel electrophoresis 21For PCR analysis of blood samples in liquid form using the SureDirect Blood PCR Kit, follow the instructions in this chapter.For analysis of dried blood samples, instead see Chapter 3 on page 23. 11Agilent Technologies2 Analysis of Liquid Blood Samples PCR primer coPBof blood that can be included in the PCR reactions by including different amounts of the blood samples in PCR reactions that amplify 12lood sample properties and sample quantity considerations• Blood samples to be analyzed with the SureDirect Blood PCR Kit in liquid form should be obtained from a standard blood collection tube containing an anticoagulant such as heparin, EDTA, or citrate. Blood collected in the presence of an anticoagulant may be stored at –80°C for later analysis, if required. Blood samples stored at –80°C should be divided into single-use aliquots and should be thawed only once.• Protocols are provided for a typical, optimized PCR assay, using 5-µL human blood samples present at 20% (v/v) of the complete PCR reaction. You can design different assay conditions, using modified blood sample volume and proportion of blood in the PCR reaction (2% to 40%) as needed for your application. Adjusting the proportion of blood in the assay requires some modification to the standard amplicon recovery procedure, as outlined in the optimization parameters and protocol steps below. • For analysis of non-human blood samples, first determine the amount purified genomic DNA as template.nsiderationsCR primer considerationsDesign PCR primers for analysis of blood samples using the following considerations:• The SureDirect Blood PCR Kit can be used to amplify a variety of target sizes and composition profiles with some optimization of conditions. For assays that require the least optimization, however, design targets in the 200 to 400 bp range and with typical GC-content. The positive control provided with the kit produces a 248-bp amplicon. • Primers should have a calculated Tm value of approximately 63°C to 73°C. You can use the IDT OligoAnalyzer 3.1 tool, provided by the recommended primer vendor, to estimate primer Tm. The recommended annealing temperature for amplification using the SureDirect Blood PCR Kit is 5°C lower than the average primer Tm.Before using the chosen primer pair for target amplification from blood samples, validate the specificity and yield of the PCR reaction using SureDirect Blood PCR Kitwell-characterized targets from plasmid DNA. Analysis of Liquid Blood Samples 2 OSureDirect Blood PCOptimization parametersptimization parametersAmplification of challenging PCR targets, including GC-rich and long (>1kb) targets, may require optimization of PCR conditions including proportion of blood in the assay, use of DMSO, and adjustment of thermal cycling parameters. Optimization for long (>1 kb) targets• Begin optimization of long target PCR by including blood samples present at 2% to 5% (v/v) of the complete PCR reaction. For the 25-µL PCR reaction volume in the following protocol, start assay optimization using 0.5 µL to 1.25 µL of blood, adding PCR-grade water to bring the volume of each sample to 5 µL. Larger volumes of blood may be included in the assay if needed for a particular target, but additional optimization steps, such as DMSO inclusion, may be required.• Amplification of some long targets may be improved by including DMSO in the PCR reaction mixture. If amplification appears inefficient, titrate DMSO in 1% increments in the range of 1% to 6% in the final PCR reaction, using the DMSO provided in the kit. Adjust the amount of water added to the reaction mixtures accordingly. DMSO may increase PCR error rates so should be avoided in cases where there is no benefit to yield or specificity.• Use the specialized cycling condtions provided in Table 8 on page 18 for long PCR targets.Optimization for GC-rich targets• Begin optimization of GC-rich target PCR by including blood samples present at 5% to 20% (v/v) of the complete PCR reaction. For the 25-µL PCR reaction volume in the following protocol, start assay optimization using 1.25 µL to 5 µL of blood, adding PCR-grade water to bring the volume of each sample to 5 µL when required. Larger volumes of blood may be tested in the assay if indicated for a particular target, but higher proportions of blood in the assay may decrease performance of the system, making detection of challenging targets more difficult.R Kit 132 Analysis of Liquid Blood Samples Optimization p14arameters• Amplification of GC-rich targets may be improved by including DMSO in the PCR reaction mixture. If amplification appears inefficient, titrate DMSO in 1% increments in the range of 1% to 8% in the final PCR reaction, using the DMSO provided in the kit. Adjust the amount of water added to the reaction mixtures accordingly. DMSO may increase PCR error rates so should be avoided in cases where there is no benefit to yield or specificity.• Use the specialized cycling condtions provided in Table 9 on page 18 for long PCR targets.SureDirect Blood PCR KitAnalysis of Liquid Blood Samples 2 SsSureDirect Blood PCStep 1. PCR-amplify the target DNA from liquid blood samplestep 1. PCR-amplify the target DNA from liquid blood amplesYou can design SureDirect PCR assays for liquid blood samples using a variety of reaction volumes and blood sample volumes, as long as the provided 2× master mix makes up 50% of the final PCR reaction volume.The example protocol below analyzes 5-µL blood samples in a 25-µL PCR reaction volume, for a final PCR reaction composition of 20% (v/v) blood. The volume of blood analyzed and the proportion of blood in the assay can be modified, if needed, by changing the reaction volume and amount of water added to the reaction mixture. Blood samples may be added to the PCR reaction at 10–40% of the final reaction volume with some modifications to post-PCR sample processing steps, as detailed in the protocol below.Example 25-?l PCR assay using 5-?l blood samples1 For runs that include the provided positive control, first dilute 1 µL of the 10× Control Primers + Template solution in 9 µL of PCR-grade water in a 1.5 mL Lo-Bind tube. Keep on ice.2 Place 5 µL of each blood sample to be PCR-analyzed in wells of 8-well strip tubes.As appropriate, include a no-template control by placing 5 µL of PCR-grade water in an additional strip tube well. Positive control reactions should be set up by placing either 5 µL of PCR-grade water (for verifying performance of kit components) or 5 µL of blood sample (to analyze effects of blood sample factors on PCR performance) in an additional strip tube well.3 Prepare a mixture of PCR master mix and PCR primers, as described in Table 5. Combine the reagents in a 1.5 mL Lo-Bind tube and keep on ice. Prepare the amount required for the number of samples and no-template controls in the run, plus excess.For positive control reactions, combine the reagents listed in Table 6 in a separate tube.R Kit 152 Analysis of Liquid Blood Samples Step 1. PCR-am16NOTE4 Mix by vortexing, then spin the tube briefly to collect the liquid.5 Transfer 20 µL of the mixture prepared in Table 5 or Table 6 to the appropriate sample wells prepared in step 2.6 Mix by vortexing and then spin the tube strips briefly to collect the liquid.7 Place the tube strips in a thermal cycler and run the PCR program appropriate program for your target:PCR-grade water 6.5 µLSureDirect Blood PCR 2X Master Mix (MM) 12.5 µL1× Control Primers + Template dilution, prepared in step 1 1 µLTotal 20 µLTable 6 Preparation of positive control reaction mixtureReagent Volume for 1 reactionplify the target DNA from liquid blood samplesTable 5 Preparation of PCR master mix + primers for multiple test reactionsReagent Volume for 1 reactionVolume for 8 reactions (includes excess)Volume for 16 reactions (includes excess)PCR-grade water 5.5 µL 46.75 µL 93.5 µLSureDirect Blood PCR 2X Master Mix (MM)12.5 µL 106.25 µL 212.5 µLForward Primer (10 µM) 1 µL 8.5 µL 17 µLReverse Primer (10 µM) 1 µL 8.5 µL 17 µLTotal 20 µL 170 µL 340 µLWhen amplifying GC-rich, long, or other difficult targets, results may be improved by adding DMSO to the PCR reaction mixture. See “Optimization parameters” on page 13 for suggested titration ranges. When adding DMSO to the mixture, decrease the amount of water added to the mixture accordingly.SureDirect Blood PCR KitAnalysis of Liquid Blood Samples 2 SureDirect Blood PCStep 1. PCR-amplify the target DNA from liquid blood samples• For typical targets, including the positive control template and primer pair included with the kit, optimize PCR starting with the program in Table 7 • For long targets (>1 kb), optimize PCR starting with the program in Table 8 • For GC-rich targets, optimize PCR starting with the program in Table 9 For all target types, do the PCR using a heated lid. If your thermal cycler has multiple ramp rate options, use the standard ramp rate; do not use a fast ramp rate setting..Table 7 Recommended PCR program for typical targetsSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 30 95°C 30 secondsPrimer Tm–5°C** Use the annealing temperature appropriate for your primer pair. For the positive control provided with the kit, use 60°C.30 seconds72°C 1 minute†† Yield of some amplicons close to 1 kb length may be improved by using a 75-second extension time.3 1 72°C 5 minutes4 1 4°C HoldR Kit 172 Analysis of Liquid Blood Samples Step 1. PCR-am18plify the target DNA from liquid blood samplesTable 8 Recommended PCR program for targets >1 kbSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 35 94°C 45 secondsPrimer Tm–5°C* 45 seconds72°C 1 minute per kb3 1 72°C 5 minutes4 1 4°C Hold* Use the annealing temperature appropriate for your primer pair.Table 9 Recommended PCR program for GC-rich targetsSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 30 98°C 30 secondsPrimer Tm–5°C** Use the annealing temperature appropriate for your primer pair.30 seconds72°C 1 minute3 1 72°C 5 minutes4 1 4°C HoldSureDirect Blood PCR KitAnalysis of Liquid Blood Samples 2 CAUTIONimmediately removed. If required, samples may be subjected to a second round of centrifugation.SureDirect Blood PCincluding the amplicons, is recovered from the cell debris in the suspension by centrifugation.Agilent recommends performing the centifugation steps below using a microcentrifuge rated at 15,000 rpm and equipped with a strip tube-compatible rotor, such as the Eppendorf 5424 benchtop microcentrifuge with strip-tube rotor accessory. Using this optimal equipment facilitates efficient recovery of the amplicon-containing supernatant from the substanial amount of cell debris released during this procedure. Alternatively, the procedure can be adapted to use a clinical centrifuge with a swinging-bucket rotor, using the modified procedure described on page 20.1 Load the strip tubes in a microcentrifuge equipped with a strip tube rotor.2 Centrifuge the strip tubes at 15,000 rpm for the time period appropriate for your sample composition, as shown in Table 10.3 Immediately after the centrifugation period is complete, collect the supernatants using a pipette, transferring the liquid from each strip tube well to separate, fresh LoBind tubes.Table 10 Recommended centrifugation timePercent Blood (v/v) in PCR Reaction Centrifugation Timeup to 10% 5 minutes10–20% 5–10 minutes20% 10 minutes20–40% 10–15 minutes40% at least 15 minutesIt is important to collect the supernatant from the cell debris immediately after centrifugation. The supernatant may be reabsorbed by the viscous pellet when not Step 2. Recover the amplicons from lysed cell debrisStep 2. Recover the amplicons from lysed cell debrisExposing the blood samples to PCR conditions produces a viscous cell lysate material. In this step, the aqueous component of the lysate, R Kit 192 Analysis of Liquid Blood Samples 20Step 2. Recover the amplicons from lysed cell debrisModified amplicon collection protocol using a clinical centrifugeA clinical centrifuge with a swinging-bucket rotor, such as the Eppendorf 5804 centrifuge, may be used for the adapted amplicon recovery procedure, as described below.1 Equip the centrifuge buckets with a 96-well plate adapter or similar.2 Load the strip tubes in the 96-well plate adapter racks.3 Centrifuge the strip tubes at 11,000 rpm for 45 minutes.4 Immediately after the centrifugation period is complete, collect the supernatants using a pipette, transferring the liquid from each strip tube well to separate, fresh LoBind tubes.CAUTION It is important to collect the supernatant from the cell debris immediately after the centrifugation step. The supernatant may be reabsorbed into the debris when not immediately removed. If required, samples may be subjected to a second round of centrifugation.SureDirect Blood PCR KitAnalysis of Liquid Blood Samples 2 SSureDirect Blood PCStep 3. Analyze the amplicons by gel electrophoresistep 3. Analyze the amplicons by gel electrophoresisAnalyze the presence of the target amplicon in the recovered supernatant using gel electrophoresis. Typical qualitative analysis conditions are electrophoresis of 10 µL of the supernatant using 1–3% agarose, or NuSieve agarose gels, depending on the PCR product size. Include a DNA ladder with fragments in the appropriate size range in one lane of each gel to verify the size of the amplicon(s) present in each sample.Expected Results for the Positive ControlAmplification of the positive control target using the provided Control Pimers + Template produces a single 248-bp amplicon. Analysis of 10 µL of the positive control reaction is expected to produce a readily-visualized band using standard laboratory gel processing protocols.Use the same 10-µL volume for analysis of postive controls run in plain water samples and for analysis of supernatants recovered from centrifugation of postive controls run in blood samples. R Kit 212 Analysis of Liquid Blood Samples Step 3. Analyz22e the amplicons by gel electrophoresisSureDirect Blood PCR KitSureDirect Blood PCR Kit Protocol3Analysis of Dried Blood SamplesPCR primer considerations 24Preparation and storage of dried blood samples 24Optimization parameters 25Step 1. PCR-amplify the target DNA from dried blood samples 26Step 2. Analyze the amplicons by gel electrophoresis 30For PCR analysis of dried blood samples using the SureDirect Blood PCR Kit, follow the instructions in this chapter.For analysis of liquid blood samples, instead see Chapter 2 on page 11. 23Agilent Technologies3 Analysis of Dried Blood Samples PCR primer coPpurified genomic DNA as template.P24reparation and storage of dried blood samples• Blood samples to be analyzed with the SureDirect Blood PCR Kit in dried form should be prepared by applying liquid blood samples to paper supports. Agilent recommends using Whatman 903 Protein Saver Cards for this purpose. • Blood samples collected with or without anticoagulant may be analyzed using the dried blood sample protocol.• Using a pipette or capillary tube, apply 50 µL of each blood sample to the center of a circular spot marked on the sample card. Avoid touching the card with the pipette or capillary tube.• After application, thoroughly dry the blood samples on the paper supports. Store the dried cards sealed in plastic bags with dessicant packets at room temperature or at –20°C.mBefore using the chosen primer pair for target amplification from blood samples, validate the specificity and yield of the PCR reaction using nsiderationsCR primer considerationsDesign PCR primers for analysis of blood samples using the following considerations:• The SureDirect Blood PCR Kit can be used to amplify a variety of target sizes and composition profiles with some optimization of conditions. For assays that require the least optimization, however, design targets in the 200 to 400 bp range and with typical GC-content. The positive control reagents provided with the kit produce a 248-bp amplicon. • Primers should have a calculated Tm value of approximately 63°C to 73°C. You can use the IDT OligoAnalyzer 3.1 tool, provided by the recommended primer vendor, to estimate primer Tm. The recommended annealing temperature for amplification using the SureDirect Blood PCR Kit is 5°C lower than the average primer T .SureDirect Blood PCR KitAnalysis of Dried Blood Samples 3 SureDirect Blood PCOCAUTIONfor long PCR targets.the PCR reaction mixture. If amplification appears inefficient, titrate DMSO in 1% increments in the range of 1% to 8% in the final PCR reaction, using the DMSO provided in the kit. Adjust the amount of water added to the reaction mixtures accordingly. DMSO may increase PCR error rates so should be avoided in cases where there is no benefit to yield or specificity.• Use the specialized cycling condtions provided in Table 15 on page 29 Optimization parameters• Just before analysis, punch out a 2-mm diameter disc containing the blood sample. Sterilize the punch tool between samples. ptimization parametersAmplification of challenging PCR targets, including GC-rich and long (>1kb) targets, may require optimization of PCR conditions including use of DMSO and adjustment of thermal cycling parameters. Optimization for long (>1 kb) targets• Amplification of some long targets may be improved by including DMSO in the PCR reaction mixture. If amplification appears inefficient, titrate DMSO in 1% increments in the range of 1% to 6% in the final PCR reaction, using the DMSO provided in the kit. Adjust the amount of water added to the reaction mixtures accordingly. DMSO may increase PCR error rates so should be avoided in cases where there is no benefit to yield or specificity.• Use the specialized cycling condtions provided in Table 14 on page 29 for long PCR targets.Optimization for GC-rich targets• Amplification of GC-rich targets may be improved by including DMSO in To reduce the risk of sample loss, do not punch the discs from the sample cards in a hood with the fan on. R Kit 253 Analysis of Dried Blood Samples Step 1. PCR-amS26plify the target DNA from dried blood samplestep 1. PCR-amplify the target DNA from dried blood samplesThe example protocol below analyzes dried blood samples on 2-mm diameter filter discs in a 25-µL PCR reaction volume. You can modify the design of the SureDirect PCR assay, including changing the reaction volume, as long as the provided 2× master mix makes up 50% of the final PCR reaction volume.1 For runs that include the provided positive control, first dilute 1 µL of the 10× Control Primers + Template solution in 9 µL of PCR-grade water in a 1.5 mL Lo-Bind tube. Keep on ice.2 Add 5 µL of PCR-grade water into wells of 8-well strip tubes, filling enough wells for the number of dried blood samples in the run.As appropriate, add 5 µL of PCR-grade water to additional, empty wells for no-template control and positive control reactions.3 Place 2-mm diameter filter discs containing the dried blood samples into the strip tube wells (one disc per well).4 Prepare a mixture of PCR master mix and PCR primers, as described in Table 11. Combine the reagents in a 1.5 mL Lo-Bind tube and keep on ice. Prepare the amount required for the number of samples and no-template controls in the run, plus excess.For positive control reactions, combine the reagents listed in Table 12 in a separate tube.SureDirect Blood PCR KitAnalysis of Dried Blood Samples 3 SureDirect Blood PCNOTE5 Mix by vortexing, then spin the tube briefly to collect the liquid.6 Transfer 20 µL of the mixture prepared in Table 11 or Table 12 to the sample wells containing blood samples in 5 µL of PCR-grade water (from step 3, above).7 Mix well by vortexing and then spin the tube strips briefly to collect the liquid.PCR-grade water 6.5 µLSureDirect Blood PCR 2X Master Mix (MM)12.5 µL1× Control Primers + Template dilution, prepared in step 1 1 µLTotal 20 µLTable 12 Preparation of positive control reaction mixtureReagent Volume for 1 reactionStep 1. PCR-amplify the target DNA from dried blood samplesTable 11 Preparation of PCR master mix + primers for multiple test reactionsReagent Volume for 1 reactionVolume for 8 reactions (includes excess)Volume for 16 reactions (includes excess)PCR-grade water 5.5 µL 46.75 µL 93.5 µLSureDirect Blood PCR 2X Master Mix (MM)12.5 µL 106.25 µL 212.5 µLForward Primer (10 µM) 1 µL 8.5 µL 17 µLReverse Primer (10 µM) 1 µL 8.5 µL 17 µLTotal 20 µL 170 µL 340 µLWhen amplifying GC-rich, long, or other difficult targets, results may be improved by adding DMSO to the PCR reaction mixture. See “Optimization parameters” on page 25 for suggested titration ranges. When adding DMSO to the mixture, decrease the amount of water added to the mixture accordingly.R Kit 273 Analysis of Dried Blood Samples Step 1. PCR-am28plify the target DNA from dried blood samples8 Place the tube strips in a thermal cycler and run the PCR program appropriate program for your target:• For typical targets, including the positive control template and primer pair included with the kit, optimize PCR starting with the program in Table 13 • For long targets (>1 kb), optimize PCR starting with the program in Table 14 • For GC-rich targets, optimize PCR starting with the program in Table 15 For all target types, do the PCR using a heated lid. If your thermal cycler has multiple ramp rate options, use the standard ramp rate; do not use a fast ramp rate setting..Table 13 Recommended PCR program for typical targetsSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 30 95°C 30 secondsPrimer Tm–5°C** Use the annealing temperature appropriate for your primer pair. For the positive control provided with the kit, use 60°C.30 seconds72°C 1 minute†† Yield of some amplicons close to 1 kb length may be improved by using a 75-second extension time.3 1 72°C 5 minutes4 1 4°C HoldSureDirect Blood PCR KitAnalysis of Dried Blood Samples 3 SureDirect Blood PCStep 1. PCR-amplify the target DNA from dried blood samplesTable 14 Recommended PCR program for targets >1 kbSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 35 94°C 45 secondsPrimer Tm–5°C* 45 seconds72°C 1 minute per kb3 1 72°C 5 minutes4 1 4°C Hold* Use the annealing temperature appropriate for your primer pair.Table 15 Recommended PCR program for GC-rich targetsSegment Number of Cycles Temperature Time 1 1 90°C 5 minutes2 30 98°C 30 secondsPrimer Tm–5°C** Use the annealing temperature appropriate for your primer pair.30 seconds72°C 1 minute3 1 72°C 5 minutes4 1 4°C HoldR Kit 293 Analysis of Dried Blood Samples Step 2. AnalyzS30e the amplicons by gel electrophoresistep 2. Analyze the amplicons by gel electrophoresisAnalyze the presence of the target amplicon using gel electrophoresis. Typical qualitative analysis conditions are electrophoresis of 10 µL of each PCR-amplified sample using 1–3% agarose, or NuSieve agarose gels, depending on the PCR product size. Include a DNA ladder with fragments in the appropriate size range in one lane of each gel to verify the size of the amplicon(s) present in each sample.Expected Results for the Positive ControlAmplification of the positive control target using the provided Control Pimers + Template produces a single 248-bp amplicon. Analysis of 10 µL of the positive control reaction is expected to produce a readily-visualized band using standard laboratory gel processing protocols.SureDirect Blood PCR Kitwww.agilent.com??Agilent Technologies, Inc. 2014Version B.0, April 2014*600920-12*600920-12In This BookThis guide contains information to run the SureDirect Blood PCR Kit protocol.Agilent Technologies Before You Begin Procedural Notes Safety Notes Reagents Required for the Protocol Contents of the SureDirect Blood PCR Kit Required Equipment for Analysis of Liquid Blood Samples Required Equipment for Analysis of Dried Blood Samples Analysis of Liquid Blood Samples PCR primer considerations Blood sample properties and sample quantity considerations Optimization parameters Step 1. PCR-amplify the target DNA from liquid blood samples Step 2. Recover the amplicons from lysed cell debris Step 3. Analyze the amplicons by gel electrophoresis Analysis of Dried Blood Samples PCR primer considerations Preparation and storage of dried blood samples Optimization parameters Step 1. PCR-amplify the target DNA from dried blood samples Step 2. Analyze the amplicons by gel electrophoresis