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2 × Rapid Taq 预混液 P222

2 × Rapid Taq 预混液 P222

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产品详细信息

应用笔记

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产品介绍

2 × Rapid Taq Master Mix contains Taq DNA Polymerase, elongation promoting factor, dNTP and an optimized buffer system. The amplification speed of this product can reach 15 sec/kb, which is suitable for rapid PCR. The maximum amplification speed within 1 kb can reach 1 sec/kb, greatly saving reaction time. The pre-prepared 2 × Master Mix only needs to add primers and templates to perform amplification, which reduces pipetting operations and improves detection throughput and results reproducibility. The kit has excellent amplification performance and high storage stability. It is suitable for PCR amplification within 5 kb using genome as template and PCR amplification within 10 kb using plasmid and λDNA as template. The protective agent added to the system allows 2 × Master Mix to maintain stable activity after repeated freezing and thawing. The kit provides a version containing electrophoresis buffer and green loading dye, which can be directly electrophoresed after the reaction that is convenient to use. The PCR product has an adenine at the 3' end, which can be directly cloned into T vectors and is suitable for ClonExpress and TOPO cloning kits (Vazyme #C112/C113/C115/C601).

产品优势

快速放大速度:15 sec/kb,1 kb内极限放大速度可达1 sec/kb;
操作方便:加入引物和模板即可进行扩增,无需冰上操作,含染料产物可直接电泳;
稳定性好:反复冻融50次,活性无明显下降

组件

P222 Components

存储

Store at -20℃  for up to 18 months; can be stored at 4℃ for 4 months after thawing

引文

Huang, Jianfa, et al. "Migration of pre-induced human peripheral blood mononuclear cells from the transplanted to contralateral eye in mice." Stem cell research & therapy 12.1 (2021): 1-13.

Wang, Tianren, et al. "MFN2 Deficiency Impairs Mitochondrial Functions and PPAR Pathway During Spermatogenesis and Meiosis in Mice." Frontiers in cell and developmental biology 10 (2022).

Cao, Qiqi, et al. "The Recurrent Mutation in PATL2 Inhibits Its Degradation Thus Causing Female Infertility Characterized by Oocyte Maturation Defect Through Regulation of the Mos-MAPK Pathway." Frontiers in cell and developmental biology (2021): 116.

常问问题

Q1: The amplification efficiency is low and there are no amplified bands in the test group.

A1: (1) Primers

Check whether the synthetic primers have degraded due to improper storage; review primer design and use BLAST to examine primer specificity or redesign primers.

(2) 模板

Long-term storage or repeated freeze-thaw cycles will lead to DNA breakage, nicking, or degradation, so the template should be the freshly prepared double-stranded DNA. An excessively high GC content in the template will make it difficult to separate DNA double strands; in this case, add PCR Enhancer (Cat. No. P021) to effectively lower the melting temperature. Crude sample templates may contain inhibitors, so it is recommended to lower the template concentration (use after dilution; if a plant leaf is used, make sure the plant is not rich in polysaccharides or polyphenols, take a fresh, young leaf, and trim it to the size of the end of a yellow pipette tip). If cDNA is used, check the purity and integrity of the RNA used for reverse transcription.

(3) Enzyme

The enzyme used in the reaction has been inactivated. Repeat the experiment with a fresh enzyme or an enzyme from another source.

(4) Amplification system

The reaction system has been prepared incorrectly. Repeat the experiment with the correct system.

(5) Reaction program

检查变性温度是否正确,PCR仪显示的温度与实际温度是否一致。 如果温度太高,酶会在前几个循环中迅速失活。 如果温度太低,模板将不会完全变性。 如果使用酵母作为模板,预变性时间可延长至 10 分钟。 如果退火温度不合适,可以通过测试退火温度的梯度来确定合适的退火温度。 如果目标片段很长,请尝试触地式 PCR 程序。 检查延长时间是否足够。

 

Q2:扩增后的条带微弱。

A2: (1) 底漆

检查合成引物是否降解。

(2) 模板

检查模板的质量。 长期保存或反复冻融循环会导致 DNA 断裂、刻痕或降解,因此应使用新鲜制备的双链 DNA 作为模板。 如果模板已用完,则使用初始扩增产物的连续稀释作为后续扩增的模板。

(3)反应程序

试试触地式 PCR 程序; 增加延伸时间和循环次数。

 

Q3:扩增特异性差,或者有非特异性产物。

A3: (1) 底漆

引物设计不是最优的。 如果引物与目标序列中的非特异性位点结合或彼此结合形成二聚体,则通过降低引物浓度或在必要时重新设计引物来进行优化。

(2) 模板

如果模板不纯或被污染,请准备新的模板。

如果模板发生降解,或模板用量过多,可通过电泳检测模板的完整性和浓度,必要时再次纯化模板。 有关模板输入量,请参阅使用说明。

(3)反应程序

反应程序不是最优的。 如果存在小于目标条带的非特异性杂条带,则提高退火温度并减少循环次数。 如果存在大于目标条带的非特异性杂条带,则减少延伸时间和循环次数。

 

Q4:扩增产物在凝胶上电泳时条带弥散或拖尾。

A4: (1) 凝胶

在准备过程中完全融化凝胶。

(2) 引物

检查引物是否降解。

(3) 模板

模板已降级或过多。 通过电泳检测模板的完整性和浓度,必要时准备新的模板。 有关模板输入量,请参阅使用说明。 如果目标条带较长,以cDNA为模板,检查反转录RNA的纯度和完整性,在不使用随机引物的情况下再次进行反转录。

(4) 反应程序

反应程序不是最优的。 如果退火温度不合适,可以通过测试退火温度的梯度来确定合适的退火温度。 试试触地式 PCR 程序。

 

Q5:空白对照(NTC)组有扩增产物。

A5: (1) 有问题的引物设计

扩增序列与非目标序列同源。 因此,非目标序列也在 PCR 中被扩增。

(2) 如果扩增产物对应的条带与目标条带大小相同,则表明存在污染。

用新鲜的 Mix、水或引物重复实验。 在超洁净工作台上准备反应系统,以尽量减少气溶胶污染。

(3) 为避免基因组或大片段对目标序列的交叉污染,请小心执行这些步骤,以防止目标序列被移入上样移液器或从离心管中溢出。

(4) 为避免空气中的小核酸片段污染目的基因,可采用巢式PCR方法减少或消除污染。

 

Q6: Do dyed PCR products affect restriction digestion?

A6: Tests show that dyed PCR products do not affect restriction digestion.

猫。 不。:
- - 请选择 - -

应用笔记摘要

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