基本描述

英文名称

DNALyse Amplification Kit

储存温度

室温,-20°C储存,避免反复冻融

运输条件

超低温冰袋运输

产品介绍

产品内容

D669986	Component	50 T	Storage
D669986A	Buffer SA	15 mL	RT
D669986B	2×PCR MasterMix	1 mL	-20℃. Avoid freeze/thaw cycle.
D669986C	Proteinase K	12.5 mg	RT
D669986D	Proteinase K Storage Buffer	1.25 mL	RT

产品简介

本试剂盒采用独特的缓冲体系，包含了快速制备基因组DNA和PCR扩增的所有试剂，适用于从各种动植物组织、细菌中一步提取基因组DNA并用于PCR扩增。整个提取过程无需液氮研磨，无需有机溶剂抽提，无需无水乙醇沉淀，提取的DNA质量稳定。本试剂盒提供的2×PCR MasterMix是一种兼容性强的PCR试剂，能够高效特异扩增DNA样品，该试剂包括DNA聚合酶、dNTPs、MgCl2、反应缓冲液、PCR反应增强剂等。具有快速简便、灵敏度高、特异性强、稳定性好等特点，特别适合于高通量的筛选。

实验前的准备及重要注意事项

1. 向Proteinase K中加入指定用量的Proteinase K Storage Buffer使其溶解，-20℃保存。配制好的Proteinase K勿长时间室温放置，避免反复冻融，以免影响其活性。

1. 样品应避免反复冻融，否则会导致提取的DNA片段较小且提取量也下降。

2. 使用前请检查Buffer SA是否出现结晶或沉淀，如有结晶或沉淀出现，请将Buffer SA于56℃水浴重新溶解。

3. 本产品提供的PCR MasterMix为2×，使用时需加入模板和引物，并加入RNase-Free Water补足体积，使其浓度为1×即可进行反应。

操作步骤

1. 取材：

植物材料：取约10 mg样本于离心管（自备）中；

动物材料：取约10 mg样本于离心管（自备）中；

细菌：取生长状态良好的菌液200-800 μL于离心管（自备）中，收集菌体。

2. 加入200 μL Buffer SA，涡旋混匀。

注意：如果是植物叶片和动物组织，应尽量用研磨杵研磨：如果是植物种子，应事先破碎并研细；细菌、1-3mm鼠尾样本可直接涡旋裂解。

3.加入10 μL Proteinase K，混匀，56℃孵育10分钟，95℃处理5分钟。

注意：1）如果为动物组织样本，可适当延长56℃孵育时间至30分钟；如有未完全消化的任何组织，应在下一步离心后尽量彻底去除。

2）95℃处理时注意不要超过5分钟。

4．13,000 rpm（~17,900×g），离心5分钟。

5. 转移上清至新的离心管（自备）中，直接用于PCR扩增，或4℃或-20℃保存溶液。

6. PCR扩增：

1）PCR反应体系：

以下举例为常规PCR反应体系和反应条件，实际操作中应根据模板、引物结构和目的片段大小不同进行相应的改进和优化。

试剂	20μL体系	终浓度
2×PCR MasterMix	10μL	1×
Forward Primer, 10 µM	1μL	0.4 μM
Reverse Primer, 10 µM	1μL	0.4 μM
Template DNA	1-2μL	/
RNase-free Water	up to 20μL	/

注意：引物浓度请以终浓度0.2-0.6μM作为设定范围的参考。扩增效率不高的情况下，可提高引物的浓度；发生非特异性反应时，可降低引物浓度，由此优化反应体系。

2）PCR反应条件：

注意：1）一般实验中退火温度比扩增引物的熔解温度Tm低5℃，退火时间一般为30-60秒，无法得到理想的扩增效率时，适当降低退火温度；发生非特异性反应时，提高退火温度，由此优化反应条件。

2）延伸时间根据所扩增的片段大小设定，本产品中所包含的Taq DNA Polymerase的扩增效率为1kb/30s 。

3）可根据扩增产物的下游应用设定循环数。循环次数太少，扩增量不足；循环次数多，错配机率会增加，非特异性背景严重。所以，在保证产物得率的前提下，应尽量减少循环次数。

3）结果检测：反应结束后取5 µL反应产物，直接进行琼脂糖凝胶电泳检测。

Product Content

D669986	Component	50 T	Storage
D669986A	Buffer SA	15 mL	RT
D669986B	2×PCR MasterMix	1 mL	-20℃. Avoid freeze/thaw cycle.
D669986C	Proteinase K	12.5 mg	RT
D669986D	Proteinase K Storage Buffer	1.25 mL	RT

Products

This kit adopts a unique buffer system containing all the reagents for rapid preparation of genomic DNA and PCR amplification, and is suitable for one-step extraction of genomic DNA from various plant and animal tissues and bacteria and for PCR amplification. The whole extraction process does not require liquid nitrogen grinding, organic solvent extraction, anhydrous ethanol precipitation, and the quality of extracted DNA is stable. The 2×PCR MasterMix provided in this kit is a highly compatible PCR reagent that can amplify DNA samples efficiently and specifically, which includes DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR reaction enhancer and so on. It is characterized by fast and easy, high sensitivity, high specificity, good stability, etc. It is especially suitable for high throughput screening.

Pre-experiment Preparation and Important Notes

1. Add the specified amount of Proteinase K Storage Buffer to Proteinase K to dissolve it and store it at -20℃. Do not leave the prepared Proteinase K at room temperature for a long time, and avoid repeated freezing and thawing to avoid affecting its activity.

2. Repeated freezing and thawing of the samples should be avoided, as this will result in smaller DNA fragments and a decrease in the amount of extracted DNA.

3. Before use, please check Buffer SA for crystallization or precipitation. If crystallization or precipitation occurs, please re-dissolve Buffer SA in a 56℃ water bath.

4. The PCR MasterMix provided with this product is 2×, when using it, you need to add template and primer, and add RNase-Free Water to make up the volume, so that its concentration is 1× to carry out the reaction.

Procedure

1. Fetch:

Plant material: take about 10 mg of sample in a centrifuge tube (provided);

Animal material: take about 10 mg of sample in a centrifuge tube (provided);

Bacteria: Take 200-800 μL of bacteria in good growth condition in a centrifuge tube (self-provided) and collect the bacteria.

2. Add 200 μL of Buffer SA and vortex to mix.

Note: In the case of plant leaves and animal tissues, they should be ground with a pestle and mortar as much as possible: in the case of plant seeds, they should be crushed and finely ground beforehand; bacterial and 1-3 mm rat-tail samples can be directly vortex lysed.

3. Add 10μL of Proteinase K, mix well, incubate at 56℃ for 10 minutes, and treat at 95℃ for 5 minutes.

Note: 1) In the case of animal tissue samples, the incubation time at 56°C may be extended to 30 minutes as appropriate; if there is any incompletely digested tissue, it should be removed as thoroughly as possible after centrifugation in the next step.

2) Be careful not to exceed 5 minutes when treating at 95°C.

4. 13,000 rpm (~17,900 x g), centrifugation for 5 minutes.

5. Transfer the supernatant to a new centrifuge tube (self-prepared) and use it directly for PCR amplification, or store the solution at 4℃ or -20℃.

6. PCR amplification:

1) PCR reaction system:

The following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.

reagents	20 μL system	final concentration
2×PCR MasterMix	10 μL	1×
Forward Primer, 10 µM	1 μL	0.4 μM
Reverse Primer, 10 µM	1 μL	0.4 μM
Template DNA	1-2 μL
RNase-free Water	up to 20 μL

Note: Please use the final concentration of 0.2-0.6μM as a reference for setting the range of primer concentration. If the amplification efficiency is not high, the concentration of primer can be increased; if a non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.

2）PCR reaction conditions:

move	temp	timing
premutability	94°C	2min
denaturation	94°C	30s
annealing (metallurgy)	55-65°C	30s 30-40 cycles
reach	72°C	60s
ultimate extension	72°C	5min

Note: 1) In general, the annealing temperature is 5℃ lower than the melting temperature of the amplification primer Tm, and the annealing time is generally 30-60 seconds. When the desired amplification efficiency cannot be obtained, the annealing temperature should be lowered appropriately; when a non-specific reaction occurs, the annealing temperature should be raised, thus optimizing the reaction conditions.

(2) The extension time is set according to the size of the fragment to be amplified, and the amplification efficiency of Taq DNA Polymerase included in this product is 1kb/30s .

3) The number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too low, the amplification is insufficient; if the number of cycles is high, the chance of mismatch will increase and the non-specific background will be serious. Therefore, the number of cycles should be minimized under the premise of ensuring the product yield.

(3) Result detection: 5 µL of reaction product was taken at the end of the reaction and directly detected by agarose gel electrophoresis.

安全和危险性（GHS）

SDS英文版

质量标准

质量标准

d669986_dnalyse amplification kit.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

C of A & Other Certificates(BSE/TSE, COO):

输入批号以搜索分析图谱:

溶液计算器

摩尔浓度计算器

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稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

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体积 1 (V1)

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浓度 2 (C2)

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复溶计算器

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快速DNA提取扩增试剂盒

库存信息

基本描述

安全和危险性（GHS）

质量标准

质检证书（CoA,COO,BSE/TSE 和分析图谱)

溶液计算器

摩尔浓度计算器

稀释计算器

复溶计算器