- Sequencing, including NGS library preparation
- Hot start PCR, multiplexing
- Direct gel loading option
- Fast high-fidelity PCR (up to 100 x Taq)
- Long PCR up to 20 kb
- Amplification of complex (GC/AT rich) templates
- Blunt-end cloning and other applications
- Hot start enzyme for increased sensitivity and great multiplex results
- Fast, high yield PCR with the fidelity 100x higher than Taq
- Up to 20 kb long PCR even from complex templates
- Increased processivity for faster amplification and higher yield
- High thermostability for better denaturation of GC rich templates
- Best choice for NGS and other sequencing applications
- Red master mix for direct gel loading, supplied with water
Derived from our HiFi polymerase, the highQu ALLin™ Mega HS HiFi DNA Polymerase provides much lower error rate PCR with a 100 higher fidelity compared to Taq. Compared to Mega HiFi, this hot start enzyme version allows for even higher sensitivity and specificity of PCR as well as for a room-temperature reaction setup and is an excellent choice for multiplex reactions. The ALLin™ Mega HS HiFi DNA Polymerase is engineered to be much faster and to generate a higher yield of long PCR products up to 20 kb from complex GC-rich templates. Therefore the ALLin™ Mega HS HiFi DNA Polymerase is an excellent choice for long and very complex PCR applications where the highest fidelity is demanded. It is an enzyme of choice for cloning and all kind of sequencing applications including NGS. Generated blunt-ended PCR products are suitable for ligation into blunt vectors. To increase ligation efficiency, the use of HighEnd™ Repair Kit (HER0101) is recommended.
The convenience of ALLin™ Mega HS HiFi DNA Polymerase is maximized by the use of 2X Red Mastermix providing the advantage of reduced pipetting and direct gel loading. The master mix is even supplied with PCR Water, and the only thing to add is the template with primers.
ALLin™ Mega HS HiFi Red Mastermix, 2X is premixed with red dye and density reagents for direct loading on the gels after the PCR. In a 2% agarose TAE gel the dye migrates with~350 bp DNA, in 1% agarose TAE gel with ~ 600 bp DNA fragments.
- 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.
- For complex, GC rich templates, use 99-100°C denaturation temperature, it might help to increase the yield.
- For established PCRs, try two-step cycling protocol with a combined annealing-denaturation step of 70°C (68°C to 75°C).
- Run an annealing temperature gradient (2°C increments) from 60°C to 66°C to choose the best conditions.
- The longer the amplicon, the longer the extension time: depending on the complexity of the template, perform extension from 10 sec/kb to 30 sec/kb. Longer extension for more complex templates is needed.
Prepare a 50 µl PCR reaction
|Rev. & For. Primers||variable, To 0.2 - 0.6 µM each (~2µl of 10 µM)|
|cDNA Template or
|<100 ng or
10 - 200 ng
|PCR Water||to 25 μl|
|ALLin™ Mega HS HiFi Red Mastermix, 2X||25 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: 60-66°C- 15 sec|
25-35 cycles: 72°C – 30 sec (10-30 sec/kb)
- After the PCR store probes shortly on ice, for long term store at -20°C.
- Load probes directly on the agarose gel. ALLin™ Mega HiFi Red Mastermix, 2X is premixed with red dye and density reagents for direct loading after the PCR.
- In a 2% agarose TAE gel the dye migrates with~350 bp DNA, in 1% agarose - with ~ 600 bp DNA.
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