2X PCR Taq Mastermix, 5.0 ml (200 PCR reactions) - Must be stored at - 20° C
Features and Benefits - Saves preparation time by combining Taq DNA Polymerse, dNTPs and reaction buffer
in a ready-to-use mixture.
- Reduces the risk of contamination by decreasing the number of pipetting steps.
- Provides consistent reaction performance and results.
The 2X PCR Taq MasterMix is a ready-to-use mixture of high quality Taq DNA
Polymerase, deoxynucleotides, and reaction buffer in a 2X concentration. It contains
all the necessary reagents for amplification of DNA along with an inert blue dye and
a stabilizer which allow direct loading of the final products onto a gel for analysis. To
set up a PCR reaction: add DNA template, primers and water. PCR products that are
amplified up to 6 kb in length with Taq DNA Polymerase contain a single base (A) 3’
overhang.
Shipping and Storage Keep at –20°C for long term storage. 2X PCR Taq MasterMix and 2X PCR Taq MasterMix
with dye are stable at 4°C for three months or for fifteen freeze-thaw cycles. For daily
use, we recommend keeping an aliquot at 4°C.
Protocol All PCR experiments should be assembled in a nuclease-free environment. In
addition, DNA sample preparation, reaction set-up and subsequent reaction(s)
should be performed in separate areas to avoid cross contamination. The use of
“clean”, automatic pipettors designated for PCR and aerosol resistant barrier tips are
recommended. Always keep the control DNA and other templates to be amplified
isolated from the other components.
A negative control reaction (omitting template DNA) should always be performed in
tandem with sample PCR to confirm the absence of DNA contamination.
1. Add the following components to a sterile 0.2 ml PCR tube sitting on ice.
| Components | Volume | Final Concentration |
| Template DNA | 100 ng | 2 ng/µl |
| Forward primer (10 µM) | 1 - 2.5 µl | 200 - 500 nM |
| Reverse primer (10 µM) | 1 - 2.5 µl | 200 - 500 nM |
| 2X PCR Taq MasterMix/ with dye | 25 µl | 1X |
| Nuclease-free H2O | up to 50 µl | - |
We recommend preparing a mastermix for multiple reactions to minimize
reagent loss and enable accurate pipetting.
2. Mix contents of tube and centrifuge briefly.
3. Incubate tube in a thermal cycler at 94°C for 3 mins to completely denature the
template.
4. Perform 30 - 35 cycles of PCR amplification as follows:
Denature: 94°C for 30 sec
Anneal: 45 - 72°C for 30 sec
Extend: 72°C for 1 min/1 kb template
5. Incubate for an additional 5 mins at 72°C and maintain the reaction at 4°C. The
samples can be stored at -20°C until use.
6. Analyze the amplification products by agarose gel electrophoresis and visualize
by ethidium bromide or SafeView™ (Cat No. G108) staining. If 2X PCR Taq
MasterMix with dye is used, load the samples directly without adding additional
loading dye. Use appropriate molecular weight standards.
