ALLin™ RPH Mastermix
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
Detailní popis
Applications
- Long PCR (up to 35 kb) with higher fidelity
- Amplification of complex (GC rich) templates
- Crude sample PCR, multiplex PCR, TA cloning
Benefits
- Long (up to 35 kb) high-fidelity (5X higher than Taq) amplification
- High yields in standard and fast PCR, on GC rich DNA and crude samples
- ALLin™ mastermix supplied with PCR Water
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.
The convenience of ALLin™ RPH Polymerase is maximized by the use of 2X Mastermix providing the additional advantage of reduced pipetting and minimized errors. The mastermix is even supplied with PCR Water, and the only thing to add is the template with primers.
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 |
| PCR Water | to 25 μl |
| ALLin™ RPH Mastermix, 2X | 25 µl |
- Mix gently, avoid bubbles
- Place into the instrument set like:
| Initial denaturation | 1 cycle: 95°C - 1-2 min |
| Denaturation | 25-40 cycles: 95°C - 15 sec |
| Annealing | 25-40 cycles: 55-65°C- 15 sec |
| Extension |
25-40 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.
| CAT. | SIZE | COMPONENTS | COMPOSITION |
| HLM0101 | 200 r of 50 µl |
5 x 1 ml - ALLin™ RPH Mastermix, 2X 5 x 1 ml - PCR Water |
1X mastermix contains 0.25 mM dNTPs, 3 mM MgCl2, enhancers, stabilizers. |
| HLM0105 | 1000 r of 50 µl |
25 x 1 ml - ALLin™ RPH Mastermix, 2X 25 x 1 ml - PCR Water |
1X mastermix contains 0.25 mM dNTPs, 3 mM MgCl2, enhancers, stabilizers. |
|
Storage |
In the dark at -20°C. |
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