ALLin™ RPH Polymerase
highQu ALLin™ RPH Polymerase (Robustní, korektura, Hot-start Polymerase) je všestranný inženýrský enzym kombinující nejlepší vlastnosti polymerázy pro dokonalost v nejnáročnějších aplikacích PCR, jako je detekce nízké kopie, dlouhá nebo vysoce věrná PCR, amplifikace komplexních templátů, hrubý vzorek PCR a multiplexování.
Polymeráza ALLin™ RPH má 5krát vyšší věrnost než polymeráza Taq DNA a produkuje produkty s A-ocáskem vhodné pro ligaci do klonovacích vektorů TA.
Pro maximální pohodlí je k dispozici ALLin™ RPH Mastermix, 2X.
Detailní popis
highQu ALLin™ RPH Polymerase (Robust, Proofreading, Hot-start Polymerase) is the versatile engineered enzyme combining best polymerase properties for excellence in most demanding PCR applications, like low copy detection, long or high fidelity PCR, amplification of complex templates, crude sample PCR and multiplexing.
ALLin™ RPH Polymerase has 5 times higher fidelity than Taq DNA Polymerase and produces A-tailed products suitable for ligating into TA cloning vectors.
For the maximum convenience the ALLin™ RPH Mastermix, 2X is available.
Important Notes
- Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
- Include a no-template control and positive control in parallel.
- Thaw and keep reagents on ice. Mix well before use.
- The longer the amplicon, the longer the extension time: 15 sec/kb - <5kb and 40 to 60 sec/kb - <5kb-35 kb. Use >90 sec extension for multiplexing.
- Run an annealing temperature gradient from 55°C to 65°C to choose the best specificity conditions.
- Do not use fast cycling for multiplexing.
Prepare a 50 µl PCR reaction
| Rev. & For. Primers | variable, up to 0.4 µM final each (2 µl of 10 µM each) |
| cDNA Template or gDNA Template |
<100 ng or 5-500 ng |
| 5X ALLin™ RPH Buffer | 10 µl |
| Water | to 49 μl |
| ALLin™ RPH Polymerase, 5 u/µl | 0.25 - 1 µl |
- Mix gently, avoid bubbles.
- Place into the instrument set like:
| Initial denaturation | 1 cycle: 95°C - 1 min |
| Denaturation | 25-35 cycles: 95°C - 15 sec |
| Annealing | 25-35 cycles: 55-65°C – 15 sec |
| Extension |
25-35 cycles: 72°C – ~10 min (for 10 kb) 15 sec/kb for <5 kb amplicons 40-60 sec/kb for >5 kb amplicons |
- After the PCR store probes shortly on ice, for long term store at -20°C.
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