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PCR Protocol for Platinum II Taq Hot-Start |
The Invitrogen Platinum II Taq Hot-Start DNA Polymerase protocol provides a reliable PCR protocol for high-specificity DNA amplification. This protocol is optimized for routine PCR, GC-rich targets, and complex templates, delivering consistent yield and sensitivity while allowing room-temperature reaction setup.
Materials required
- Platinum II Taq Hot-Start DNA Polymerase
- Template DNA (cDNA, genomic DNA, plasmid DNA, phage DNA)
- Forward and reverse primers
- Invitrogen 10 mM dNTP Mix
- Water, nuclease-free
- 0.2- or 0.5-mL nuclease-free microcentrifuge tubes
Package contents of Platinum II Taq Hot-Start DNA Polymerase
- Platinum II Taq Hot-Start DNA Polymerase
- 5X Platinum II PCR Buffer (provides 1.5 mM MgCl2 at 1X)
- Platinum GC Enhancer (optional)
Storage: Store all components at –20°C.
Kit sizes: 100 reactions, 500 reactions, 2500 reactions.
Platinum II Taq DNA polymerase protocol steps
1. Thaw, mix, and briefly centrifuge all components before use.
2. Determine the correct amount of water required to reach your final reaction volume. For a 50 µL reaction mix, prepare PCR master mix by adding the following components to each tube. Mix and then briefly centrifuge components.
| Component | Volume | Final concentration |
|---|---|---|
| Water, nuclease-free | to 50 µL | — |
| 5X Platinum II PCR Buffer | 10 µL | 1X |
| 10 mM dNTP mix | 1 µL | 0.2 mM each |
| Platinum GC Enhancer (optional) | 10 µL | 1X |
| Platinum II Taq Hot-Start DNA Polymerase | 0.4 µL | 0.04 U/µL |
Tips:
- When using Platinum II Taq DNA polymerase, it is not necessary to perform the PCR set up on ice.
- For multiple reactions, prepare a master mix of components common to all reactions to minimize pipetting error, then dispense the appropriate volumes into each 0.2-mL or 0.5-mL PCR tube before adding template DNA and primers.
- GC Enhancer is recommended for targets with >65% GC content.
Notes:
- Mg2+: Platinum II PCR Buffer provides 1.5 mM MgCl2 in the final reaction concentration. If the primers and/or the template contain chelators such as EDTA or EGTA, the apparent Mg2+ optimum may be shifted to higher concentrations.
- We recommend a final primer concentration of 0.2 µM, but this can be varied in the range of 0.1–0.5 µM for each primer.
- If possible, design the primers with one or two G and C bases at the 3’ end.
3. Add template DNA and primers. Cap tube, mix, then briefly centrifuge contents.
Maximum template per 50 µL reaction:
- Genomic DNA: 0.5–500 ng per 50 µL reaction
- Plasmid/viral DNA: 1 pg–50 ng
- cDNA reaction: 1–5 µL per 50 µL PCR
- Do not exceed 500 ng total DNA per reaction
| Component | Volume | Final concentration |
|---|---|---|
| 10 µM forward primer | 1 µL | 0.2 µM |
| 10 µM reverse primer | 1 µL | 0.2 µM |
| Template DNA | varies | <500 ng/rxn |
4. Run a thermal cycler program set to the following parameters according to the protocol to be performed.
- 2-step protocol: For simple amplicons ≤1 kb with 45–65% GC sequences
- 3-step protocol: For longer, GC-rich, and complex amplicons or cDNA targets
| Step | 3-step protocol | 2-step protocol | |||
|---|---|---|---|---|---|
| Temperature | Time | Temperature | Time | ||
| Initial denaturation | 94°C | 2 minutes | 94°C | 2 minutes | |
| 25–35 PCR cycles | Denature | 98°C | 15 seconds | 98°C | 5 seconds |
| Anneal | 60°C | 15 seconds | 60°C | 15 seconds | |
| Extend | 68°C | 15 seconds/kb | |||
| Hold | 4°C | hold | 4°C | hold | |
5. Use your PCR product immediately in down-stream applications, or store it at –20°C.
Product formats
| Stand-alone enzyme | 2X master mix | |
|---|---|---|
| Platinum II Taq Hot-Start DNA Polymerase | Platinum II Hot-Start PCR Master Mixes (colorless) | Platinum II Hot-Start PCR Master Mixes (green) |
Flexibility to optimize reaction conditions |
Download protocol |
Download protocol |
FAQs on PCR protocol using Platinum II Taq DNA Polymerase
For most primers designed using standard primer design rules, the recommended annealing temperature is 60°C.
If amplification is suboptimal:
- Perform a gradient PCR to fine-tune the annealing temperature
- Consider redesigning primers if nonspecific bands persist
- 2-step protocol: Recommended for simple amplicons ≤1 kb with 45–65% GC content
- 3-step protocol: Recommended for longer amplicons, GC-rich targets, complex templates, and cDNA targets
If experiencing low yield with the 2-step protocol, switch to the 3-step protocol.
Platinum II Taq extends 1 kb per 15 seconds at a 68°C extension temperature.
- Use 15 seconds per kb at 68°C extension temperature
- Extension time can be prolonged up to 1 minute/kb without negatively affecting specificity. This allows longer and shorter amplicons to cycle together in the same protocol.
Recommended troubleshooting actions:
- Annealing temperature too low → Increase above 60°C or run gradient PCR
- Too much template DNA → Reduce input (<500 ng recommended)
- Too many cycles → Use 25–35 cycles as recommended
- Poor primer design → Redesign primers if necessary
Recommended troubleshooting actions:
- Ensure proper initial denaturation (94°C for 2 minutes) to activate the hot-start enzyme
- Check primer and template concentrations
- Check primer design
- Use fresh high-quality dNTPs
- Use 3-step protocol
- Increase the number of cycles
Platinum II Taq has template-independent terminal transferase activity, adding a single 3′ A overhang to PCR products. This is useful for TA cloning applications. An end-repair step is required if PCR products will be used in blunt-end cloning.
For excellent performance:
- Design primers 18–30 bases long
- Aim for 40–60% GC content
- Target a Tm between 55–65°C
- Avoid primer pairs with >10°C Tm difference
- Avoid self-complementary primers and primer pairs complementary at 3’ ends
- If possible, include 1–2 G or C bases at the 3’ end
- Verify specificity using sequence alignment tools
When these rules are followed, most primers will anneal specifically at 60°C in Platinum II PCR buffer. Use primer design programs such as the Invitrogen OligoPerfect Designer primer design tool.
| Feature | Platinum II Taq DNA polymerase | Standard Taq DNA polymerase |
|---|---|---|
| Hot-start | Antibody-mediated hot-start mechanism, which blocks enzyme activity at room temperature and restores activity during the initial denaturation step (94°C) | No |
| Room temperature setup | Yes | High risk of nonspecific products |
| Inhibitor resistance | Enhanced | Standard |
| Optimized 60°C annealing | Yes | No |
| Extension speed | ~1 kb/15 seconds | Typically slower |
| Maximum amplicon length | Up to 5 kb | Similar, but often less robust |
仅供科研使用，不可用于诊断目的。
Stylesheet for Classic Wide Template adjustments