ข้อมูลจำเพาะ
แทค ดีเอ็นเอ โพลีเมอเรส 1000 U with mixed buffer (Shipping with icebag)
พื้นที่จัดเก็บ Conditions:
Taq DNA Polymerase and Taq พีซีอาร์ Kits should be stored at -30 to -15°C in a constant-temperature freezer upon receipt.
Test:
Notes Before Starting:
- Provided with Load PCR Buffer containing gel-loading reagent and gel-tracking dyes.
- PCR Buffer and Load PCR Buffer yield a final concentration of 1.5 mM MgCl2 in the reaction mix. Adjust Mg2+ concentration if necessary by adding 25 mM MgCl2.
- High-quality PCR-grade dNTP Mix (10 มม) is available separately if needed.
- Keep PCR tubes on ice until placed in the thermal cycler.
- Include a No Template Control (NTC) in every assay.
Preparation and Mixing:
- Thaw buffers and reagents at room temperature or on ice, then keep them on ice after complete thawing.
- Prepare a reaction mix containing all PCR components except template DNA. Prepare 10% more volume than required.
- Mix the reaction mix thoroughly and dispense it into PCR tubes.
- Add template DNA (≤1 µg/reaction) to PCR tubes. For RT-PCR, add an aliquot from the reverse transcriptase reaction, not exceeding 10% of the final PCR volume.
Reaction setup using SENO Taq DNA Polymerase
| ส่วนประกอบ | Volume/reaction | Final concentration |
| Reaction mix | ||
| 10x PCR Bufferˢ¹ or | 10 มล | 1x |
| 10x Load PCR Buffer(optional) | ||
| dNTP mix (10 mM of each) | 2 มล | 200 µM of each dNTP |
| Primer1 | Variable | 0.1–0.5 µM |
| Primer2 | Variable | 0.1–0.5 µM |
| แทค ดีเอ็นเอ โพลีเมอเรส | 0.5 มล | 2.5 units/reaction |
| RNAse-free water | Variable | |
| แม่แบบดีเอ็นเอ (added at step 4) | Variable | ≤1 µg/reaction |
| ปริมาตรปฏิกิริยาทั้งหมด | 100 µlˢ² |
Thermal Cycling:
Program thermal cycler according to manufacturer’s instructions. Refer to the provided cycling program.
Optimized cycling conditions for Taq DNA Polymerase
| Step | เวลา | อุณหภูมิ | ความคิดเห็น |
|---|---|---|---|
| Initial denaturation | 3 นาที | 94องศาเซลเซียส | |
| 3-step cycling: | |||
| การเสียสภาพ | 0.5–1 min | 94องศาเซลเซียส | |
| การหลอม | 0.5–1 min | 50–68°C | Approximately 5°C below Tm of primers. |
| ส่วนขยาย | 1 นาที | 72องศาเซลเซียส | For PCR products longer than 1 กิโลไบต์, use an extension time of approximately 1 min per kb DNA. |
| Number of cycles | 25–35 | ||
| Final extension | 10 นาที | 72องศาเซลเซียส |
Simplified Hot Start:
Start PCR program. Once the thermal cycler reaches 94°C, place PCR tubes in the cycler for improved specificity.
Post-PCR:
- After amplification, samples can be stored at 2–8°C overnight or at -20°C for longer storage.
- PCR products can be directly loaded onto agarose gel using Load PCR Buffer without prior addition of loading buffer and gel-tracking dyes. Refer to the provided table for migration distance and gel tracking dyes.
Migration distance of gel tracking dyes in Load PCR Buffer
| % TAE (TBE) กgarose gel | Red dye | Orange dye |
| 0.8 | 500 (270) bp | ~80 (<10) bp |
| 1.0 | 300 (220) bp | ~40 (<10) bp |
| 1.5 | 250 (120) bp | ~20 (<10) bp |
| 2.0 | 100 (110) bp | <10 (<10) bp |
| 3.0 | 50 (100) bp | <10 (<10) bp |
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