The Hieff™ Blood Advanced Direct PCR Kit is a kit that can directly amplify whole blood samples without DNA purification or sample pretreatment, and is compatible with fresh blood, chilled (frozen) blood, and commercial dried blood stains containing conventional anticoagulants such as EDTA, heparin, citrate, etc. The kit contains a genetically engineered DNA polymerase combination with high fidelity and resistance to PCR inhibitors, 2× Hieff™ Blood Advanced PCR Buffer with strong elongation factor, amplification enhancement factor, and optimized buffer system to efficiently amplify 8 kb of genomic fragments under rapid extension conditions, and set 3-5 sec/kb extension time for fragments below 2 kb, thereby significantly shortening the time for blood sample identification and testing. Specifications Fidelity (vs. Taq) 1 × Hot Start Yes Overhang 3'-A Polymerase Taq DNA Polymerase (Cat: 10726) Components No. Name 10188ES20 10188ES60 10188-A 2× Hieff™ Blood Advanced PCR Buffer 500 μL 2.5 mL 10188-B Hieff™ Advanced High-Fidelity DNA Polymerase Mix 20 μL 100 μL 10188-C Positive control primer mix (10 μM each) 100 μL 200 μL 10188-D 10× DNA loading buffer 1 mL 1 mL Instructions Recommended PCR reaction systems. Components Volume（μL） ddH2O to 50 μL 2× Hieff™ Blood Advanced PCR Buffer 25 μL Forward Primer (10 μM) 2 μL Reverse Primer (10 μM) 2 μL Hieff™ Advanced High-Fidelity DNA Polymerase Mix 1 μL Blood sample X 1. Mix well the components before use.2. A final concentration of 0.4 μM per primer is recommended, too high will result in non-specific amplification.3. The optimal whole blood template concentration ranges from 0.5% to 20%, and the recommended use is 10% as the initial trial condition, that is, 5 μL of whole blood is added as a template to the 50 μL reaction system, taking care to avoid aspirating blood clots. For dried blood stains stored on Whatman® filter cards, approximately 1 mm2 of round pieces of paper with blood stains can be taken and amplified directly into the PCR reaction without pretreatment. Reaction program Cycle step Temp. Time Cycles Initial denaturation 95℃ 5 min 1 Denaturation 95℃ 15 sec 30-35 Annealing 60℃ 15 sec Extension 72℃ 3-10 sec/kb Final extension 72℃ 5 min 1 The annealing temperature is a generic Tm value or 1-2°C below the primer Tm value. If the amplification product is less specific, an annealing temperature gradient can be established to find optimal annealing conditions. Extension time of 10 sec/kb can amplify most of the target fragments below 8 kb, and 3-5 sec/kb can amplify most fragments below 2 kb. If the amplification efficiency is low, the time can be appropriately extended to 20-30 sec/kb, and should not exceed 60 sec/kb. Figures Notes 1. It is recommended that the blood template be used as 10% of the total reaction volume, that is, 5 μL of whole blood is added to the 50 μL reaction system as the template, taking care to avoid aspirating blood clots.2. Extension time of 10 sec/kb can amplify most of the target fragments below 8 KB, and 3-5 sec/kb can amplify most of the fragments below 2 KB. If the amplification efficiency is low, the time can be extended to 30 sec/kb.3. After the PCR reaction, it is recommended to centrifuge the reaction product at 4000 rpm (1000× g) for 1-3 min to precipitate blood cell fragments, and take the supernatant for downstream analysis.4. This product cannot be directly used for medical diagnosis.5. For your safety and health, please wear a lab coat and wear disposable gloves.
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