PCR Taq

• Taq DNA聚合酶的功能是什么？
  1. PCR
  2. 平末端3'端加A尾
  3. 引物延伸
  4. DNA标记反应
• tcy8722太阳集团的Taq DNA聚合酶具有哪些特性？
  1. 高稳定性
  2. 高PCR产量
• 使用Taq DNA聚合酶或Green Taq DNA聚合酶时，推荐的酶用量是多少？

  0.5 U或更多

• Taq酶和Green Taq酶有什么不同？

  Green Taq酶中含有经过特殊稳定剂处理的常规Taq酶。这种Green Taq酶能够在室温（RT）条件下保持稳定，有效期长达两周。

• Taq DNA聚合酶或Green Taq是否可以用来扩增GC含量高的PCR产物？

  不可以。

• Taq DNA Polymerase（Taq酶）可以在其他缓冲液中使用吗？

  Taq DNA聚合酶在tcy8722太阳集团提供的Taq Buffer中可表现出百分之百的活性。此外，Taq DNA聚合酶同样也能很好地适用于其他缓冲液体系。为了您的方便，tcy8722太阳集团还特别提供了名为10x Taq Buffer的产品 (Cat. No. B0005)。

• 如果我想控制PCR反应中镁离子（Mg2+）的浓度，应使用哪种缓冲液？镁离子（Mg2+）的存在是否会抑制PCR反应？

  这是一个潜在的问题。因此，我们建议使用无Mg的Taq DNA聚合酶(Cat. No. E00008)。

• 如何配置使用Taq DNA聚合酶进行PCR扩增反应？

  以下是50 μl PCR反应的指导原则：

  1. 0.5 μl Taq DNA聚合酶
  2. 1 μl 10 mM dNTP
  3. 1 μl 每种引物
  4. 2 μl 基因组模板（浓度可达100 ng/μl）
  5. 5 μl 10x Taq Buffer（含有Mg2+ 的标准Taq酶缓冲液）
  6. 39.5 μl 无菌或过滤的H2O
  7. 变性在94°C
  8. 延伸1 minute/kb
• 常规PCR反应中各组分适宜的浓度是怎样的？
  1. 对于3 kb以下的片段，我们推荐在50 µl反应体系中使用1.25单位Taq DNA聚合酶。
  2. 对于特殊应用，包括扩增3–8 kb大小的片段，推荐在50 µl反应体系中使用2.5单位Taq DNA聚合酶。
• tcy8722太阳集团的Taq DNA聚合酶能够扩增的最大产物长度是多少？

  可达8 kb λDNA。

• 由Taq DNA聚合酶进行引物延伸反应或PCR反应所产生的DNA末端类型是什么？

  Taq DNA聚合酶产生的PCR产物末端为平末端，适用于TA克隆等技术。

• 为什么在琼脂糖凝胶电泳中观察不到产物？

  潜在原因可能包括PCR体系设置或程序不正确，或者电泳过程存在问题。

• 产品序列与预期序列不完全匹配。如何改进？

  尝试使用高保真DNA聚合酶。

• 在何时应当使用Taq DNA聚合酶进行引物延伸反应或PCR？

  当PCR反应开始时或在变性步骤之后，可以使用Taq DNA聚合酶。

• Taq DNA聚合酶的5'→3' flap内切酶活性是否会降解引物？

  不会，Taq DNA聚合酶的5'→3'外切酶活性只会降解在其延伸DNA片段过程中遇到的双链DNA。如果该酶在同一条链上遇到次级引物（例如用于突变体构建的引物），它会降解这种次级引物。

• 您可以在何处寻求帮助解决PCR问题？

  请发送邮件至product@genscript.com.cn.

  问题	原因	解决方法
  微量或无扩增产物	GC含量丰富的模板	在PCR反应中添加5% DMSO、1 M甜菜碱或两者都可以，以改善结果
  	模板降解	在琼脂糖凝胶上分析模板，以检查是否存在潜在的降解迹象
  	PCR抑制剂污染	使用已建立的PCR体系对模板进行测试 重复模板纯化步骤以去除残余酒精、酚或其他污染物
  	循环条件	改变退火温度 增加循环次数 延长延伸时间
  	引物问题	两条引物的浓度必须相同。最终浓度应在0.2 µM ~ 0.6 µM之间 确保引物未降解
  	酶浓度低	每次增加0.5 U的聚合酶用量
  	MgCl2浓度低	每次增加0.25mM的MgCl2浓度
  	退火温度不合适	提高退火温度
  	模板浓度不合适	对模板进行系列稀释测试
  	引物二聚体形成	当PCR循环温度达到94°C时，插入PCR反应管并继续进行程序

PCR克隆试剂盒

• GenBuilder™试剂盒与PCR克隆试剂盒有何区别？

  GenBuilder™无缝克隆技术提供了高效、快速且直接的克隆方法，无需经过传统克隆中常见的限制酶切割、连接以及有时所需的磷酸化等繁琐步骤。该技术适用于任何DNA序列和任何载体，唯一的要求是PCR引物需包含与载体序列至少15个核苷酸同源的部分。

• 技术信息在手册中并未提供，这是为什么？

  目前，我们正在等待GenBuilder的专利批准。一旦生效，手册中将提供更详细的信息说明。

• 当GenBuilder™反应进行转化后，为什么没有得到足够多的菌落？

  这可能由四个因素引起。

  1. 转化感受态细胞的转化效率较低。
  2. 使用的反应混合物过多。
  3. PCR DNA或线性化载体中含有抑制性污染物。
  4. 载体与插入片段的摩尔比不合适。
     请参阅手册中的故障排除部分。
• 当GenBuilder™反应进行转化后，为什么会得到不正确的菌落？

  这可能由两个因素引起。

  1. 克隆载体未能完全线性化。
  2. 克隆反应被具有相同抗生素抗性的质粒污染。
• What factors need to be considered when designing the primer?

  Two sets of primers are used to amplify the gene of interest:

  1. 15-bp homology regions to the vector, flanking the cloning site into the insert.
  2. Specific gene sequence. If the sequence has 5' overhang, the start of the 15 bp homology must begin from the 5' most extension to include the overhang; if the sequence has a 3' overhang, homology should begin where the DNA becomes double-stranded. Please refer to the manual for illustration of primer design.
• What should the purity of my primer be to be compatible with the GenBuilder™ cloning method?

  Desalted oligos (from a qualified supplier) are suitable for cloning with the GenBuilder™ PCR cloning Kit.

• Can multiple fragments be cloned into a single vector using this kit?

  Yes. GenBuilder™ PCR Cloning Kit can be used for multiple fragment recombination if there are not many repeated sequences among multiple fragments. However, we recommend recombination one fragment at a time. Several performances of recombining 2 - 3 fragments demonstrated efficiency as low as 20%-30%, and lead to a direct repeat deletion.

• Can I use GenBuilder™ PCR Cloning Kit with my own vectors?

  Yes. GenScript's GenBuilder™ PCR Cloning Kit has been successfuly tested with several commercial and non-commercial vectors.

• What factors need to be considered when using GenBuilder™ PCR Cloning Kit with customers' vector?

  We recommend linearizing your vectors before using GenBuilder™ PCR Cloning Kit. Once linearized, the columned and gel-purified vector is ready for GenBuilder™ reaction.

• Will the GenBuilder™ reaction work more efficiently if I use primers that contain a longer than 15 base region of homology?

  A 15-20 bp homology is recommended.

• What is the stability of the enzyme included in GenBuilder™ PCR Cloning Kit?

  The liquid enzyme should be stored at -20°C for at least 12 months without activity loss.

  Problem	Probable Cause	Solution
  Few or no colonies are obtained from the transformation.	The competent cells have low transformation efficiency.	Check the transformation efficiency. Competent cells with >1×108 cfu/μg are recommended.
  	Too much reaction mixture is used.	Do not add more than 20 μl of reaction mixture to 50 μl of competent cells. Too much reaction mixture inhibits the transformation.
  	There are inhibitory contaminants from PCR DNA or from linearized vector. The molar ratio of vector to insert is off.	Both the PCR DNA and the linearized vector should be purified. Usually an insert/vector molar ratio of 2:1 is optimal. If the insert is as large as the linearized vector, a molar ratio of 1:1 can also be used.
  	Too long or short a recombination time.	It is recommended to keep the recombination procedure within 30 min.
  	The linearized cloning vector or primer is large.	Connect the product to the target step recombinant vector.
  	The cloning vector is not completely linearized.	Gel-purify the linearized vector.
  	The cloning reaction is contaminated with plasmids having the same antibiotic resistance.	Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.
  Most of the colonies contain no insert.	The cloning vector is not completely linearized.	Gel-purify the linearized vector.
  	The cloning reaction is contaminated with plasmids having the same antibiotic resistance.	Purified PCR DNA may contain the template plasmid, so gel-purify the PCR DNA.

质粒纯化

• 为什么我的质粒DNA产量较低?
  1. 检查细菌培养是否正确。
  2. 检查细菌细胞是否充分悬浮。
  3. 将洗脱缓冲液在30-60°C下孵育，这有助于提高产量。
• QuickClean II质粒小提试剂盒是否可用于哺乳动物细胞中质粒DNA的提取？

  不可以，该试剂盒提供了一种快速、简便且经济高效的质粒小提方法，适用于1–5毫升大肠杆菌过夜培养物。

• 对于质粒小提试剂盒，推荐使用何种培养基？

  LB培养基

• 如何确定我的质粒是高拷贝数类型还是低拷贝数类型？

  例如，一系列PUC载体是高拷贝数质粒。您通常可以从质粒名称中了解到它们的不同类型。

• 如何解决质粒提取中的基因组DNA污染问题？

  如果质粒提取过程中出现基因组DNA污染，在加入裂解缓冲液后，应轻轻倒转离心管。

• 是否可以使用QuickClean Miniprep试剂盒提取低拷贝质粒和黏粒？

  该试剂盒可以纯化低拷贝质粒，但对于黏粒则不太适用。

• tcy8722太阳集团不同试剂盒中名称相同的缓冲液是否一致？

  是的，tcy8722太阳集团名称完全相同的缓冲液在化学成分上是一致的，可以在不同试剂盒之间互换使用。例如，QuickClean II PCR纯化试剂盒中的洗涤液和洗脱缓冲液与QuickClean II PCR凝胶提取试剂盒中的洗涤液和洗脱缓冲液完全相同。

• QuickClean II PCR纯化试剂盒是否可以从实时PCR反应中去除荧光染料？

  是的，QuickClean II PCR纯化试剂盒能够有效地从实时PCR反应中去除SYBR Green染料。该试剂盒还能移除用于PCR DNA标记的荧光标记dNTP，例如Cy3-dUTP等。

• Is it necessary to repeat the wash procedure?

  The DNA eluted from the column is pure enough for most downstream applications. However, if the downstream applications are sensitive to salt carryover, it is better to wash the column twice prior to elution.

• Can I use TE buffer or water to elute DNA from the column?

  Elution Buffer is 2.5 mM Tris-HCl pH 8.5. TE buffer or water can also be used, but yield will be slightly lower. The pH of the elution solution is critical; buffers with a higher pH such as 8.5 or above will be more efficient to elute DNA from the column.

• What additional consumables does the user need?

  Ethanol is needed to prepare the wash solution for all three kits. Isopropanol is also needed for QuickClean II PCR or Gel Extraction Kit.

• Can I extract and purify DNA from gels using TAE running buffer?

  Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from gels using either TBE or TAE as the electrophoresis buffer.

• Can I extract and purify DNA from low melting point (LMP) Agarose gels?

  Yes. QuickClean II PCR or Gel Extraction Kit can be used to extract and purify DNA from low melting point (LMP) gels.

  Problem	Solution
  Obtain no plasmid DNA	If there is no plasmid DNA in the elution buffer, please check whether the ethanol had been added to wash buffer according to the volume marked on bottle label.
  Low plasmid DNA yields	Please check if the bacteria was cultured properly. Please check if the bacteria cells were resuspended completely. 3. Incubate the Elution Buffer at 30～60°C，to help increase the yields.
  Absorbance problem	Absorbance is the difference of the sample from the blank, please use the Elution Buffer to adjust the blank to a zero value and use it to dilute the sample. If the ratio of OD260/ OD230 is too low, wash the spin column for one more time. In the case of a low ratio of OD260-320/ OD280-320, there may be protein contamination. In this case please add Neutralization Buffer, and then centrifuge buffer with sufficient rotating speed, thus to make precipitation compact; be careful to pipette supernatant and avoid pipetting the precipitate. If the ratio of OD260-320/ OD280-320 is too high, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.
  Electrophoresis problem	If there is genomic DNA in the result, invert the tube gently (step 3 and 4). If there is RNA in the result, add more RNase A to Resuspension Buffer to a final concentration 100 μg/ml.