Platinum SuperFi II DNA 聚合酶是一种工程酶,具有高持续合成能力,并增强对 PCR 抑制剂的耐受性。它也可以实现快速循环方案和扩增长靶标(长达 20 kb)。Platinum 热启动技术使用专有抗体,可在 PCR 变性步骤之前抑制酶活性,防止非特异性扩增和引物降解。该技术还可在室温下进行反应构建,提高了灵敏度和得率。
由于 SuperFi II PCR 缓冲液的独特成分,按照一般设计规则设计的大多数引物对的退火温度为 60°C。缓冲液中的等稳定化分子可提高退火步骤中引物-模板的双重稳定性,并可增强特异性,而无需优化每个引物对的退火温度。使用 Platinum SuperFi II DNA 聚合酶,可以使用同一方案将不同的 PCR 测定共循环,该方案即使用通用引物退火温度和为待扩增的最长片段选择的延伸步骤。
为提高便利性,我们提供 Platinum SuperFi II 预混液和 Platinum SuperFi II DNA 聚合酶,并提供即用型混合物和 SuperFi II PCR 缓冲液和 dNTP,因此减少了 PCR 反应构建过程中的移液步骤数量。也可以使用 Platinum SuperFi II Green PCR 预混液,此外,其还包含一种密度试剂和两种示踪染料,用于 PCR 产物直接上样到凝胶上,进一步从设置到最终分析简化了 PCR 的工作流程。
I'm having a hard time amplifying/cloning low copy cDNAs via PCR. Do you have any suggestions for me?
You may want to consider using our Platinum SuperFi II DNA Polymerase (Cat. No. 12361010) and try optimizing your PCR conditions if needed. The Platinum SuperFi II DNA Polymerase demonstrates a superior detection sensitivity with as little as 0.4 ng of genomic DNA. It is also a good idea to include a positive control reaction in parallel to your test PCR to ensure there are no issues with your reagents during handling or use over time. Please note that control templates may be ordered through our GeneArt Gene Synthesis (de novo sequence with 100% guarantee delivered in a vector) or Strings (up to 3 Kb, blunt-ended dsDNA pools, cloned into ZeroBlunt TOPO vector, convenient for multiple controls) services.
What are your recommendations when working with long amplicons using Platinum SuperFi II DNA Polymerase?
Good lab practices are important for long fragment amplification. These include using high-quality templates (pure, fresh, and intact) and fresh primer solutions. Reducing primer concentration to 0.2 µM may also improve the results.
Can I use Platinum SuperFi DNA Polymerase cycling protocol and primer annealing temperature with Platinum SuperFi II DNA Polymerase?
Yes, Platinum SuperFi II DNA Polymerase works at both universal 60 degrees C annealing temperature and at annealing temperatures calculated with a Tm calculator.
Can I perform TA cloning with Platinum SuperFi II DNA Polymerase?
Platinum SuperFi II DNA Polymerase produces blunt-ended PCR products that can be cloned directly into blunt-ended cloning vectors. TA cloning is also possible if 3' dA-overhangs are added after PCR. We recommend purifying the PCR products of Platinum SuperFi DNA Polymerase before adding the overhangs. The procedure for adding 3' dA-overhangs (TA cloning) includes the following steps:
- Purify the PCR product (e.g., with a PCR purification kit or phenol extraction/DNA precipitation). Before adding the overhangs, PCR product must be purified, as the strong proofreading activity of any remaining Platinum SuperFi DNA Polymerase will degrade the added 3' dA-overhangs.
- Perform 3' dA addition with a Taq DNA polymerase.
Reaction components:
Purified PCR product
0.2 mM dATP
1x Taq Buffer
1 U Taq DNA Polymerase
Incubate the reaction for 20 min at 72 degrees C.
- Proceed to TA cloning. For optimal ligation efficiency, we recommend using fresh PCR products, since 3' dA-overhangs can be lost during storage