用于高灵敏度、准确的长片段PCR扩增

  • 扩增得到长片段PCR产物(最长至40 kb DNA)
  • 高保真酶确保低错配率
  • 独特的缓冲液体系最大程度的减少PCR条件优化
  • 扩增低拷贝和高GC含量模板

QIAGEN LongRange PCR Kit提供专用的、高度可靠的解决方案,用于对基因组DNA和cDNA,甚至是低拷贝数模板,进行长片段扩增。新型的高保真酶可确保低错配率。该试剂盒包含核苷酸,高度适用于复杂的基因分型实验。

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QIAGEN LongRange PCR Kit (20)
For 20 x 50 µl reactions: LongRange PCR Enzyme Mix (40 U), LongRange PCR Buffer, 5x Q-Solution, RNase-Free Water, 10 mM dNTPs
206401
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QIAGEN LongRange PCR Kit (100)
For 100 x 50 µl reactions: LongRange PCR Enzyme Mix (200 U), LongRange PCR Buffer, 5x Q-Solution, RNase-Free Water, 10 mM dNTPs
206402
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QIAGEN LongRange PCR Kit (250)
For 250 x 50 µl reactions: LongRange PCR Enzyme Mix (500 U), LongRange PCR Buffer, 5x Q-Solution, RNase-Free Water, 10 mM dNTPs
206403
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The QIAGEN LongRange PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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High yields of very long PCR products.|Protection of DNA from excessive damage.|
Long PCR products (27 kb and 24 kb) were amplified from human genomic DNA using the QIAGEN LongRange PCR Kit and 2 additional long-range PCR kits from the indicated suppliers, according to the manufacturer's instructions. The QIAGEN LongRange PCR Kit provided much higher yields of specific product than the other kits that were tested.|Genomic DNA from leukocytes was pretreated in water (Water), long-range PCR buffer from Supplier A (Standard buffer), or QIAGEN LongRange PCR Buffer (LongRange Buffer), and then directly used for PCR () or incubated at 95°C (+) before PCR. Amplification reactions (50 μl) were performed using the QIAGEN LongRange PCR Kit. M: marker. Pretreatment of DNA in QIAGEN LongRange PCR Buffer provided protection from damage, resulting in increased yield. Reduced product yields were achieved due to greater DNA damage when DNA was pretreated in water or standard long-range PCR buffers.|
Performance

QIAGEN LongRange PCR Kit包含所有需要用于长片段PCR扩增(长达40 kb)的组分。独特的耐热DNA聚合酶具有增强的处理能力,甚至能够从极小量的模板中扩增长片段的PCR产物。酶混合物具有高延伸率和校对提高保真度的能力(比Taq DNA聚合酶至多高5倍的保真度)。新型的缓冲体系甚至能够从复杂的基因组DNA中,进行高度特异性长片段PCR扩增(参见"High yields of very long PCR products")。此外,该缓冲液能提供保护防止脱嘌呤、确保全长度扩增(参见"Protection of DNA from excessive damage")。在LongRange PCR Buffer中预孵育的DNA的PCR产物产量,与未受损的对照DNA相似,表明缓冲体系的保护功能为长片段PCR产物扩增提供了理想的反应环境。独特LongRange PCR Buffer优化了高温下(72–94°C)pH值缓冲,降低了pH导致的模板降解。与Q-Solution(也包含在该试剂盒中)结合使用,能更进一步方便的扩增富含GC和其他“困难”的模板。

QIAGEN LongRange Kit规格
规格  长片段PCR LongRange 2Step RT-PCR
应用 克隆、测序、标准PCR可达40 kb 克隆、基因表达分析、标准两步法RT-PCR可达12.5 kb
酶组分 DNA聚合酶、高保真酶 逆转录酶、DNA聚合酶、高保真酶
所需最小优化 独特的缓冲液体系和优化的实验方案 独特的缓冲液体系和优化的实验方案
总RNA量 50 ng–4 µg总RNA
总DNA量 50–500 ng
扩增富含GC片段的模板 提供Q-Solution 提供Q-Solution
Principle

QIAGEN LongRange PCR Kit将强大的聚合酶混合物与新型的专用于有效扩增长达40 kb靶的缓冲体系结合。该试剂盒也包含Q-Solution,一种PCR添加剂有助于扩增富含GC片段的模板和dNTP。

LongRange PCR Enzyme Mix

LongRange PCR Enzyme Mix是高纯度重组耐热DNA聚合酶和持续合成能力增强因子的混合物,克隆在E. coli内。这种独特的酶混合物确保了高延伸率和高保真率,能够成功扩增极长的片段。

优化的缓冲液体系,用于极长的PCR产物

扩增大于4 kb长的PCR产物通常失败,并通常需要优化长度的操作流程。失败的原因包括非特异性引物退火或欠佳的循环条件。避免DNA损伤,如DNA脱嘌呤,在扩增长片段PCR产物尤为重要,因为模板中一个DNA损伤就足以影响聚合酶效率。PCR循环过程中的DNA损伤可使用特殊的缓冲液将其降到最低,用来稳定反应中的pH值。QIAGEN LongRange PCR Buffer经优化用于扩增长达40 kb的PCR产物,并提供有效保护防止其脱嘌呤(参见"Protection of DNA from excessive damage")。

扩增稀少和困难模板

独特的LongRange PCR Buffer优化了高温下(72–94°C)pH缓冲的性能、降低了pH导致的模板降解。与Q-Solution(已提供)相结合,能进一步优化富含GC片段和其他“困难”模板的扩增。

Procedure

QIAGEN LongRange PCR Kit包含预优化的实验方案,用于可靠的精确长片段PCR,片段长达40 kb。该试剂盒能成功运用于扩增长片段靶(所有靶都在0.1–10 kb范围内)、极长片段靶(基因组DNA >10 kb;游离态DNA 10–40 kb)、从逆转录反应获得的长cDNA模板和富含GC片段和其他“困难”模板。

Applications

QIAGEN LongRange PCR确保对标准或复杂模板(甚至低丰度或富含GC 片段的模板)进行准确的长片段扩增,并高度适用于所有PCR应用,包括测序或定点重测序、拷贝数变异的验证(CNV)、染色体易位断点图谱和其他结构变异以及克隆。

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Kit Handbooks
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For reliable and accurate long-range PCR up to 40 kb
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