USAGE NOTES Little/no product detected: Assay Design – Endpoint PCR: • Reagent was omitted from or improperly • Amplicon length: amplicons up to 4000 bp added to the assay. Verify complete adherence may be amplified. to protocol steps. Further optimization • Primer concentration: 100 nM is suitable for most of PCR protocol may be required. assays. May be varied between 50 – 1000 nM. • Check melt temperature of primers and lower the annealing temperature if needed. Template Preparation: • Improper qPCR channel selection. • Compatible templates: cDNA, gDNA, and plasmid. Ensure channel assignment matches probe. 990 Richard Ave, Suite 110 No purification is required for use of cDNA Santa Clara, CA 95050 with the Sahara Hot Start PCR Master Mix. Inconsistent replicate amplifications: United States • Purified DNA is ideal as PCR inhibition is avoidable. • Pipetting error. Ensure proper technique. Inhibition may occur with crude preparations • Insufficient mixing during preparation. www.chaibio.com despite inhibition resistance by the mix, Ensure thorough mixing of original resulting in a higher limit of detection. reagents. Ensure PCR reaction contents [email protected] • A range of 50 ng – 2 pg is typical for large genomes are adequately mixed following setup. +1 (800) 642-4002 Toll-free (i.e. human, mouse). The range of input DNA for +1 (650) 779-5577 International smaller genomes is greater. Efficiency out of range (< 90% or > 110%) Sahara Hot Start • GC content: Sahara Hot Start PCR Master Mix is or unexpected R² value: compatible with GC content from 40 – 70%. • Pipetting or dilution error during assay setup. PCR Master Mix • qPCR interference due to bubbles. Cloning: • Suboptimal reaction conditions. Chai™ and Sahara™ are trademarks of • Template concentration: may be increased • Inappropriate instrument threshold setting. Catalog #: R02151 Chai Biotechnologies Inc. up to 1000 ng to generate maximum product. For research use only • 3’ adenine overhang: a 3’ dA overhang is generated Amplification of No Template Control (NTC): by the , allowing amplified products • Reagent contamination by DNA template. Store at: to be ligated using TA cloning. Excessive formation: -20 ºC long-term • Suboptimal primer concentration or design. 4 ºC for 6 months TROUBLESHOOTING Redesign assay using software to check for www.chaibio.com Inconsistent plasmid amplifications: Avoid repeated freeze/thaw cycles primer dimer formation across all assays. Protect from light 002111 Rev A • Supercoiling of targets. Linearize the plasmid. INTRODUCTION PROTOCOL Reaction Setup: Thermocycling Protocol – Two-Step: Sahara Hot Start PCR Master Mix is a 2X mix for Prepare Master Mix: • Determine total volume required for reactions + 10%. Recommended for Real-Time PCR with endpoint PCR or quantitative probe-based detection • Thaw completely at room temperature. • Prepare assay mix for all components melt temperatures ≥ 60 ºC. using singleplex Real-Time PCR. -based Mix contents by inversion or pipetting, except template. STEP TEMP. TIME hot start technology, unparalleled thermal stability, or by gently vortexing for < 3 s. • Aliquot reaction mix into PCR tubes/plate. compatibility with targets from an expansive range • Centrifuge to collect the solution at the • Add DNA template to PCR tubes/plate. Initial Denaturation 95 ºC 1 min of organisms, and high amplification efficiency bottom of the tube. • Close tubes/seal plate. Denature 95 ºC 15 s make it an ideal mix for routine PCR applications. • Centrifuge briefly Reaction Composition – Endpoint PCR: Extend 60 ºC 30 s Cycle 30 - 45x HIGHLIGHTS COMPONENT 20 μL REACTION FINAL CONC. Thermocycling Protocol – Three-Step: • Retains stability even under extreme conditions: Sahara Hot Start 10 μL 1X Recommended for endpoint PCR, or Real-Time PCR three months at 25 °C or eight days at 50 °C, USAGE NOTES PCR Master Mix with melt temperatures < 60 ºC. followed by 20 freeze/thaw cycles Assay Design – Real-Time PCR: • Aptamer-based hot start system reduces Forward & Reverse Primers variable 50 – 1000 nM STEP TEMP. TIME • Amplicon length: amplicon lengths of 80 – 200 bp produce optimal reaction efficiency values. non-specific amplification and primer dimers Template DNA variable < 1000 ng Initial Denaturation 95 ºC 1 min • Annealing temperature: a 60 °C annealing • Amplifies up to four kb from genomic DNA, Nuclease-free Water to 20 μL Denature 95 ºC 15 s and GC-rich targets with up to 70% GC-content and extension temperature is recommended for NOTE: Based on 20 μL reaction, adjust accordingly for other volumes. Anneal 45 – 68 ºC 15 – 30 s two-step Real-Time PCR. Perform three-step SPECIFICATIONS PCR if the T of the primer is < 60 °C. Extend 72 ºC 1 kb/min m Reaction Composition – Real-Time PCR: • Primer concentration: 400 nM is suitable for Polymerase Taq DNA Polymerase Final Extension 72 ºC 5 min most assays. Primer concentration should COMPONENT 20 μL REACTION FINAL CONC. GC-Rich Performance ≤ 70% Cycle 30 - 45x be varied to achieve optimum PCR assay Sahara Hot Start 10 μL 1X Max Amplicon Length 4 kb efficiencies (90 – 110%) for the target. PCR Master Mix • Probe concentrations: 250 nM is suitable for Hot Start Yes—Aptamer Forward & Reverse Primers variable 50 – 1000 nM most targets. For high abundance targets: Supported Probes TaqMan/Hydrolysis, Molecular Beacon, Probe variable 100 – 250 nM optimize the reaction using probe concentrations Scorpions ranging from 100 – 250 nM. Template DNA variable < 100 ng Supported Templates Genomic DNA, cDNA, Plasmid DNA Nuclease-free Water to 20 μL Continues on other side

NOTE: Based on 20 μL reaction, adjust accordingly for other volumes.