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NGS Library Prep kits

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Phanta EVO HS Super-Fidelity DNA Polymerase

Phanta Super-Fidelity DNA Polymerase is a new generation of ultra-fidelity DNA polymerase modified from Pfu DNA Polymerase. It has extremely high amplification efficiency and wide template adaptability, and can be used in almost all PCR reactions. With the genetic engineering of Pfu DNA Polymerase, the processivity of Phanta Super-Fidelity DNA Polymerase has been greatly improved. Even for very complex templates, the amplification reaction can be completed accurately and quickly. Phanta EVO HS Super-Fidelity DNA Polymerase is an upgraded version of Phanta HS Super-Fidelity DNA Polymerase. Compared with the previous generation product, Phanta EVO HS has added a unique extension factor and deeply optimized the reaction system to further improve its amplification stability, fidelity and ability to amplify long fragments. Using simple templates such as lambda DNA and plasmids, Phanta EVO HS can effectively amplify fragments up to 40 kb; using complex templates such as genomic DNA, Phanta EVO HS can also effectively amplify fragments up to 20 kb. Its amplification mismatch rate is 1/100 of that of ordinary Taq polymerase and 1/12 of Pfu polymerase; and the amplification speed can reach 15 seconds/kb. In addition, Phanta EVO HS has good resistance to PCR inhibitors and can be used for bacteria, fungi, Direct PCR of plant tissue, animal tissue or whole blood samples. Strict fidelity performance and excellent amplification efficiency make Phanta EVO HS the first choice for high-fidelity PCR. Phanta EVO HS has added two monoclonal antibodies that can inhibit its 5'→3' polymerase activity and 3'→5' exonuclease activity at room temperature, enabling highly specific Hot Start PCR . The enzyme has 5'→3' polymerase activity and 3'→5' exonuclease activity, and the amplified product is blunt-ended, which is suitable for ClonExpress rapid cloning kit (C112/C113). 5 × EVO Buffer has good adaptability to PCR amplification of simple or complex templates, short fragments or long fragments. It already contains 10 mM Mg2+, you can use the MgCl2 or PCR Enhancer provided with the enzyme to optimize the reaction system.    Super fidelity: the fidelity is 53 times that of Taq DNA Polymerase and 6 times that of Pfu DNA Polymerase. Every 300,000 bases amplified, with less than 5 mismatches; Faster amplification: the limit speed can reach 0.5 sec/kb, 30 sec/kb can efficiently amplify most fragments; Longer amplification: the effective amplification length of simple templates such as plasmid and λ DNA can reach 40 kb, the effective amplification length of cDNA can reach 10 kb, and the effective amplification length of genome can reach 20 kb; Wide adaptability: It is suitable for the amplification of various GC content fragments, has super tolerance to PCR inhibitors, and can be used for direct PCR of a variety of samples.   Store at -20°C
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Phanta Super-Fidelity DNA Polymerase is a new generation of ultra-fidelity DNA polymerase modified from Pfu DNA Polymerase. It has extremely high amplification efficiency and wide template adaptability, and can be used in almost all PCR reactions. With the genetic engineering of Pfu DNA Polymerase, the processivity of Phanta Super-Fidelity DNA Polymerase has been greatly improved. Even for very complex templates, the amplification reaction can be completed accurately and quickly.
Phanta EVO HS Super-Fidelity DNA Polymerase is an upgraded version of Phanta HS Super-Fidelity DNA Polymerase. Compared with the previous generation product, Phanta EVO HS has added a unique extension factor and deeply optimized the reaction system to further improve its amplification stability, fidelity and ability to amplify long fragments. Using simple templates such as lambda DNA and plasmids, Phanta EVO HS can effectively amplify fragments up to 40 kb; using complex templates such as genomic DNA, Phanta EVO HS can also effectively amplify fragments up to 20 kb. Its amplification mismatch rate is 1/100 of that of ordinary Taq polymerase and 1/12 of Pfu polymerase; and the amplification speed can reach 15 seconds/kb. In addition, Phanta EVO HS has good resistance to PCR inhibitors and can be used for bacteria, fungi,
Direct PCR of plant tissue, animal tissue or whole blood samples. Strict fidelity performance and excellent amplification efficiency make Phanta EVO HS the first choice for high-fidelity PCR. Phanta EVO HS has added two monoclonal antibodies that can inhibit its 5'→3' polymerase activity and 3'→5' exonuclease activity at room temperature, enabling highly specific Hot Start PCR . The enzyme has 5'→3' polymerase activity and 3'→5' exonuclease activity, and the amplified product is blunt-ended, which is suitable for ClonExpress rapid cloning kit (C112/C113).
5 × EVO Buffer has good adaptability to PCR amplification of simple or complex templates, short fragments or long fragments. It already contains 10 mM Mg2+, you can use the MgCl2 or PCR Enhancer provided with the enzyme to optimize the reaction system. 

 

Super fidelity: the fidelity is 53 times that of Taq DNA Polymerase and 6 times that of Pfu DNA Polymerase. Every 300,000 bases amplified, with less than 5 mismatches;
Faster amplification: the limit speed can reach 0.5 sec/kb, 30 sec/kb can efficiently amplify most fragments;
Longer amplification: the effective amplification length of simple templates such as plasmid and λ DNA can reach 40 kb, the effective amplification length of cDNA can reach 10 kb, and the effective amplification length of genome can reach 20 kb;
Wide adaptability: It is suitable for the amplification of various GC content fragments, has super tolerance to PCR inhibitors, and can be used for direct PCR of a variety of samples.

 

Store at -20°C

We could not find any corresponding parameters, please add them to the properties table

Q1:扩增效率低,实验组无扩增条带。

A1:(1) 引物

检查人工合成的引物是否因存储条件不当而降解;引物设计是否合理,可利用BLAST检查引物特异性或重新设计引物。

(2) 模板

长期放置、反复冻融会导致模板断裂、开环或降解,应使用新鲜制备的DNA双链作为模板;模板GC含量过高会导致DNA的双链无法打开,此时加入PCR Enhancer(货号P021),可以有效降低解链温度;模板的AT含量过高,可尝试使用针对高AT的产品(货号P501/P511);模板为粗品,存在抑制物,建议降低模板浓度(稀释使用;若样本为植物叶片,要确保植物为非多糖多酚植物,取新鲜幼嫩的叶片,并将叶片面积剪小,如黄枪头尖部面积大小);若模板为cDNA,要确认逆转录所用RNA的纯度及完整度。

(3) 酶

反应所用的酶因保存或运输不当而失活,建议更换新酶或用另一来源的酶重新实验。

(4) 扩增体系

反应体系配制错误,建议重复实验。

(5) 反应程序

检查变性温度是否准确,PCR仪指示温度与实际温度是否相符,如果温度过高,酶在前几个循环就迅速失活,温度过低则模板变性不彻底;若模板为酵母菌,可将预变性时间延长10min;退火温度不合适,可对退火温度设置梯度,摸索最佳的退火温度;如果目的片段较长,可尝试Touch Down 程序;检查延伸时间是否充足。

(6) Mg2+浓度不合适

Mg2+浓度过低可影响PCR产量甚至使PCR扩增失败;Mg2+浓度过高会降低PCR的特异性,应适当调整Mg2+浓度。

Q2:扩增的条带亮度不太亮。

A2:(1) 引物

检查人工合成的引物是否因存储条件不当而降解。

(2) 模板

首先确认模板的质量,长期放置、反复冻融会导致模板断裂、开环或降解,应使用新鲜制备的DNA双链作为模板;如果模板没有了,可用首次扩增产物按倍比稀释后作为模板进行二次扩增。

(3) 酶

可适当提高酶的使用量。

(4) 反应程序

可尝试使用Touch Down程序;延长延伸时间,提高循环数。

Q3:扩增特异性差,非特异性扩增。

A3:(1) 引物

引物设计不够优化。引物与靶序列有非特异性互补或自身聚合成二聚体,可降低引物浓度进行优化,必要时重新设计引物。

(2) 模板

模板不纯,被污染,需重新制备模板。

模板降解或过量,通过电泳检查模板完整性及浓度,必要时重新纯化模板。模板的使用量请参考说明书。

(3) 反应程序

反应程序不够优化。如果出现比目的条带小的杂带,可通过提高退火温度,降低循环数调整;如果出现比目的条带大的杂带,可缩短延伸时间、降低循环数。

(4) 酶

酶量加入过多。高保真系列:50μl体系加1U。

Q4:扩增产物跑胶条带弥散或拖尾。

A4:(1) 胶

制胶时要使胶完全融化。

(2) 引物

检查人工合成的引物是否因存储条件不当而降解。

(3) 模板

模板降解或过量,可通过电泳检查模板完整性及浓度,必要时重新制备模板。模板的使用量请参考说明书。如果目的条带较长,模板是cDNA,要确认逆转录所用RNA的纯度及完整度,逆转录时不加随机引物重新逆转录。

(4) 反应程序

反应程序不够优化。退火温度不合适,可对退火温度设置梯度,摸索最佳的退火温度;尝试Touch Down 程序。

(5) 酶

酶量加入过多。高保真系列:50μl体系加1U。

Q5:空白对照出现扩增产物。

A5:(1) 引物设计不合理

扩增序列与非目的扩增序列有同源性,PCR也可以扩增出非靶序列的序列。

(2) 若扩增产物条带大小与目的条带一致,说明有污染

更换新的Mix、水或引物重复实验。反应体系在超净工作台内配制,减少气溶胶污染。

(3) 为了避免靶序列受到整个基因组或大片段的交叉污染,操作时应小心轻柔,防止将靶序列吸入加样枪内或溅出离心管外。

(4) 为了避免靶基因受到空气中小片段核酸污染,可用巢式PCR方法减轻或消除污染。

Q6:高保真酶扩增保真性差,存在突变。

A6:高保真酶在PCR时扩增产物有突变,可以从以下几点来分析:

(1) 模板本身存在突变

检查模板,若模板是质粒,建议送质粒去测序;若模板是cDNA,建议重复实验,如果仍有突变,可能是cDNA有问题,建议重新制备cDNA,制备cDNA前检测RNA的完整度及纯度。

(2) 模板反复冻融

重新制备模板。

(3) 模板长时间在紫外光下照射,引入突变

切胶回收过程中避免长时间在紫外光下照射。

(4) 基因序列与NCBI上的不一致

建议再重复一次送去测序,若结果和上一次一样,说明序列可能和NCBI上的不一致。

(5) 少量序列存在非严谨序列

有相似重组序列,导致重组错位。

Q7:条带大小与理论不符。

A7:(1) 实验体系污染

建议使用全新的试剂和枪头,对工作台进行清洁。

(2) 模板或引物使用错误

建议更换模板和引物。

(3) 存在基因亚型

建议对研究的基因进行序列分析和BLAST研究。

Q8:扩增产物跑胶时加样孔很亮,目的条带很暗。

A8:可能是循环数过多一些非特异性条带、蛋白、目的条带混合在一起形成大分子聚合物跑不下来。可向PCR产物中加入SDS,95℃加热5min,再跑胶。也可扩大体系,多扩几管,最后切胶一起回收。

Q9:逆转录后的产物cDNA作为模板,使用P505,一次应该加多少体积?

A9:说明书建议cDNA模板使用量为1-5μl(不超过PCR反应总体积的1/10),可在这个范围内尝试不同的使用量,摸索最佳使用量。

Q10:高保真酶扩增完的产物是否带A,若后面要做TA克隆该如何进行?

A10:高保真酶扩增完的产物不带A,可尝试使用Vazyme无缝克隆产品C112/C113/C115;若后面做TA克隆可用普通的Taq酶进行加A反应。

具体加A方法:在10μl体系中,加入纯化后的PCR产物1-7μl,同时加入1μl 10×Taq Buffer(含MgCl2)、0.5μl 10mM dNTPs、0.2μl Taq DNA聚合酶(不加引物),最后用超纯水补足体系,72℃反应10-15min即可。

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