Authors
highQu
Summary
Despite a big choice of different PCR enzymes and mixes, amplification of long and complex targets remains a challenging task.
We present a universal enzyme, a robust proofreading hot start polymerase, that enables fast and efficient amplification of complex and long templates.
Image 1 Introduction
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.
Materials
1. ALLin™ RPH Polymerase, 5 u/µl supplied with ALLin™ RPH Buffer that contains 0.25 mM dNTPs, 3 mM MgCl2, enhancers, stabilizers.
dNTPs are included in the reaction buffer, so you do not need to add them separately!
2. Template DNA
3. PCR-grade Water
4. PCR Cycler
Procedure
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 - shall be used for <5kb amplicons; and 40 to 60 sec/kb - shall be used for 5kb-35 kb amplicons. 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 by combining following components:
a. Rev. & For. Primers - variable, up to 0.4 µM final each (2 µl of 10 µM each)
b. cDNA Template - <100 ng
or gDNA Template - 5-500 ng
c. 5X ALLin™ RPH Buffer - 10 µl
d. Water - to 49 μl
e. 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
After the PCR, store probes on ice for an immediate use, for long term store keep them at -20°C.
Anticipated results
- Long (up to 35 kb) high-fidelity (5X higher than Taq) amplification
- High yields in standard and fast PCR, on GC rich DNA and with crude samples
Further details
Download the ALLin™ RPH Polymerase User Manual
References
http://www.highqu.com/products/pcr-qpcr/high-fidelity-long-pcr/allintm-rph-polymerase-5-u-l.html
Documents for download
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