ALLin™ Taq DNA Polymerase has the same PCR accuracy like Taq DNA Polymerase, 4.5 x 104 (a number of correct nucleotides incorporated before the error occurs) and produces A-tailed products suitable for ligating into TA cloning vectors.
The convenience of ALLin™ Taq DNA Polymerase is maximized by the use of 2X Red Mastermix providing the additional advantage of reduced pipetting and minimized errors.
ALLin™ Red Taq 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.
The mastermix is even supplied with the PCR Water, and the only thing to add is the template with primers.
- 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 - <6kb and 1 sec/kb - <1kb. 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
|<100 ng or
5 - 500 ng
|PCR Water||to 25 μl|
|ALLin™ Red Taq Mastermix, 2X||25 µl|
- Mix gently, avoid bubbles.
- Place into the instrument set like:
|Initial denaturation||1 cycle: 95°C - 1 min|
|Denaturation||40 cycles: 95°C - 15 sec|
|Annealing||40 cycles: 55-65°C - 15 sec|
|Extension||40 cycles: 72°C – 1 - 90 sec (15 sec/kb)|
Load probes on the agarose gel. The red loading dye is included in the mastermix.
ALLin™ Red Taq 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.
- Store probes on ice shortly, store at -20°C for long term.
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