HotStar HiFidelity Polymerase Kit

用于高灵敏度,高保真热启动PCR
  • 高灵敏度、高特异性
  • 独特的UA/TA克隆
  • 保真性比Taq DNA Polymerase高10倍
  • 可在室温下构建反应体系,酶活化速度快
经优化的即用型试剂盒包括高保真PCR所需的所有试剂、酶、缓冲液和dNTPs。HotStar HiFidelity DNA Polymerase是一种经特异修饰的产生A突出末端的热启动校对酶,能够直接、高效的进行UA/TA克隆。缓冲液中包含Factor SB,可在PCR体系建立时防止引物和模板的降解,提供高特异性和高保真PCR。此外,Q-Solution能够有效扩增难扩增的模板(如富含GC的模板)。
Product Product no. Cat. no. List price:
 
 
Show details
    varies
Can't order online?
To place an order via phone, email or for requesting a quote, please provide the product’s name, number and catalog number.
Product Cat. no. List price:
HotStar HiFidelity Polymerase Kit (100)
100 units HotStar HiFidelity DNA Polymerase, 5x HotStar HiFidelity PCR Buffer (incl. dNTPs), 5x Q-Solution, 25 mM MgSO4, RNase-Free Water
202602
Inquire
HotStar HiFidelity Polymerase Kit (1000)
1000 units HotStar HiFidelity DNA Polymerase, 5x HotStar HiFidelity PCR Buffer (incl. dNTPs), 5x Q-Solution, 25 mM MgSO4, RNase-Free Water
202605
Inquire
The HotStar HiFidelity Polymerase Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
  • Main Image Navi
Efficient TA/UA cloning.|Highly sensitive and reliable PCR.|Amplification of difficult templates.|Increased specificity of primer annealing.|
A 955 bp fragment was amplified from 100 ng of human genomic DNA using the HotStar HiFidelity Polymerase Kit and standard Taq DNA Polymerase. PCR products (4 µl each) were cloned using a commercially available TA or UA cloning kit. Blue-white screening was used to determine the cloning efficiency.|PCR was performed using HotStar HiFidelity DNA Polymerase (QIAGEN) and 4 high-fidelity PCR enzymes from the indicated suppliers. Parallel reactions were performed following the suppliers' recommendations, using 10 ng and 1 ng human genomic DNA. Two different high-fidelity enzymes from Supplier I were tested. A 2.3 kb fragment of the human IL9R gene was amplified in 40 PCR cycles. M: Markers.|Two different primer–template systems were amplified in duplicate using QIAGEN PCR Buffer and Taq DNA Polymerase in the absence () or presence (+) of 1x Q-Solution. Q-Solution enables specific amplification of difficult templates. [A] human angiotensin receptor II gene; [B] mouse protein kinase C gene; M: markers.|Ammonium and potassium cations in QIAGEN PCR Buffers increase specificity of primer annealing. K+ binds to phosphate groups (P) on the DNA backbone, stabilizing the annealing of the primers to the template. NH4+, which exists both as the ammonium ion and as ammonia under thermal-cycling conditions, can interact with the hydrogen bonds between the bases (B), destabilizing the weak hydrogen bonds at mismatched bases. The combined effect of the two cations maintains a high ratio of specific-to-nonspecific primer-template binding over a wide temperature range.|
Performance

HotStar HiFidelity Polymerase Kit包含一种经特异修饰的热启动校对酶,可在PCR体系建立时防止引物和模板的降解,提供高保真PCR,无需优化。通过检测,HotStar HiFidelity DNA Polymerase Kit可确保高特异性和高灵敏度扩增(参见"Highly sensitive and reliable PCR")。HotStar HiFidelity DNA Polymerase的保真性比Taq DNA polymerase高10倍。新型的HotStar HiFidelity PCR Buffer包含专有的Factor SB,提高PCR的灵敏度和可靠性,尤其在起始模板量较少的条件下。该试剂盒同时提供Q-Solution,可进一步优化PCR(参见"Amplification of difficult templates")。

HotStar HiFidelity DNA Polymerase特性

浓度:2.5 units/μl 
5'–>3'外切酶活性:否 
3'–>5'外切酶活性:
额外添加A(末端转移酶活性):
半衰期:95°C条件下>4小时
核酸酶污染:
蛋白酶污染:
RNase污染:

Principle

通常利用高保真酶3'→5'核酸外切酶的活性去除错配的整合碱基。但是,该活性会在PCR体系建立和起始时降解引物,而导致非特异性启动、扩增,甚至扩增产物失败,尤其在模板量较少的条件下。

HotStar HiFidelity DNA Polymerase

HotStar HighFidelity DNA Polymerase是从新型Pyrococcus spp.中特异分离的,经过化学修饰使聚合酶和核酸外切酶失活。5分钟热启动激活酶组分,使PCR起始阶段就有高校对能力。独特的修饰确保体系建立过程中引物和酶的完整性,防止错误启动。HotStar HiFidelity DNA Polymerase的可靠性能确保高特异性和高灵敏度的扩增。

HotStar HiFidelity PCR Buffer

新型的HotStar HiFidelity PCR Buffer包含独特的PCR添加剂,以及浓度已经优化的dNTPs和MgSO4。Factor SB是一种特有的PCR添加剂,提高PCR的灵敏度和可靠性(参见"Highly sensitive and reliable PCR")。新型的Factor SB可确保模板DNA的完整性,该DNA常常会被具有3'→5'核酸外切酶活性的高保真酶降解。

预优化试剂能够可靠的扩增特异性PCR产物,且出错率低。该缓冲液通过提高每个PCR扩增退火过程特异性引物的结合比例,确保PCR每个循环特异性扩增(参见"Increased specificity of primer annealing")。独特的KCl和(NH4)2SO4平衡组合,使得HotStar HiFidelity PCR Buffer与传统的PCR缓冲液相比在很宽的温度和Mg2+浓度范围内都有严格的引物退火条件和高度的特异性,无需进行耗时的优化。

HotStar HiFidelity PCR Buffer也含有浓度经特定优化的dNTPs,用于方便的建立PCR反应体系和获得可靠的结果。该缓冲液最大程度上增加了HotStar HiFidelity DNA Polymerase的保真性,确保低出错率。HotStar HiFidelity Polymerase Kit灵敏度高、特异性强以及使用HotStar HiFidelity PCR Buffer而无需优化的特性使其适合多种高保真PCR应用。

Q-Solution

HotStar HiFidelity Polymerase Kit同时提供Q-Solution,一种新型的PCR添加剂,通过更改DNA熔解行为促进难扩增模板的扩增。这种独特的试剂可进一步优化由模板引起的表现不理想的PCR,如过多的二级结构和富含GC的模板(参见"Amplification of difficult templates")。不同于DMSO和其他PCR添加剂,Q-Solution的浓度是确定的,没有毒性,并且保证PCR的纯度。而且PCR中加入Q-Solution不会影响PCR的准确性。

完整的试剂盒规格

HotStar HiFidelity Polymerase Kit包含PCR所需的所有组分。无需额外购买其他试剂或缓冲液。

Procedure

HotStar HiFidelity操作流程高效,只需按照试剂盒中已优化的试验方案操作即可。HotStar HiFidelity DNA Polymerase经过化学修饰,可以暂时性的使聚合酶和3'–>5'核酸外切酶失活。这种抑制作用可使引物和模板在PCR体系建立和循环起始阶段过度降解。95°C条件下孵育5分钟即可激活聚合酶和校对活性,可以整合入已有的热循环程序。这种修饰确保可在室温下方便的建立反应体系,而不会影响PCR的特异性和产量。

TA/UA克隆

与其他高保真DNA聚合酶相比,使用HotStar HiFidelity DNA Polymerase获得的PCR产物可直接用于TA-或UA-克隆(参见"Efficient TA/UA cloning")。

产物的长度和灵敏度

鉴于HotStar HiFidelity Polymerase Kit的高灵敏度,即使使用极微量的起始原料也可扩增片段(如1 ng的人基因组DNA)。使用基因组DNA获得的产物长度可达5 kb,而λDNA获得的产物长度可达10 kb,表明HotStar HiFidelity Polymerase Kit适合多种应用,包括用于克隆的长片段DNA的扩增。

Applications

HotStar HiFidelity Polymerase Kit适合多种要求高灵敏度和低出错率的热启动PCR应用,如克隆和定点突变。

Feature
Specifications
Applications Cloning, mutation analysis, TA- or UA-cloning procedures
Enzyme activity 3' -> 5' exonuclease activity, A-addition
Mastermix No
Reaction type PCR amplification, proofreading
Real-time or endpoint Endpoint
Sample/target type Genomic DNA and cDNA
Single or multiplex Single
With/without hotstart With hotstart

You are not authorized to download the resource

Brochures & Guides
2
Kit Handbooks
1
For highly sensitive and reliable high-fidelity hot-start PCR
Show details