基本描述

英文名称

RNApure Plant Kit（DNase I）

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

R669988	Component	50T	Storage
R669988A	DNase I	1000 U	-20℃. Avoid freeze/thaw cycle.
R669988B	10×Reaction Buffer	1000 μL	-20℃. Avoid freeze/thaw cycle.
R669988C	Buffer RL	35 mL	RT
R669988D	Buffer RLC	35 mL	RT
R669988E	Buffer RW1	40 mL	RT
R669988F	Buffer RW2 (concentrate)	11 mL	RT
R669988G	RNase-Free Water	10 mL	RT
R669988H	Spin Columns FL with Collection Tubes	50 sets	RT
R669988I	Spin Columns RM with Collection Tubes	50 sets	RT
R669988J	RNase-Free Centrifuge Tubes (1.5 mL)	50 EA	RT

产品简介

本试剂盒用于从各种植物中提取纯化高品质总RNA，也适用于真菌菌丝RNA的提取。独特的分离柱，用于匀质化和过滤高粘度的植物或真菌裂解物，同时采用硅基质膜吸附RNA进行纯化，使多聚糖等各种污染物通过洗涤被有效去除，经洗脱的RNA可直接用于各种下游实验。由本试剂盒提取RNA分子量大于200碱基，纯度高，几乎无DNA残留。如果是对微量DNA非常敏感的RNA实验，残留的DNA可利用无RNase的DNase在柱上进行消化去除。提取的RNA可用于Northern Blot、Dot Blot 、RT-PCR和体外翻译等实验。

自备试剂：β-巯基乙醇、无水乙醇（新开封或提取RNA专用）。

实验前准备及重要注意事项

1. 预防RNase污染，应注意以下几方面：

1）使用无RNase的塑料制品和枪头，避免交叉污染。

2）配制溶液应使用无RNase的水。

3）操作人员戴一次性口罩和手套，实验过程中要勤换手套。

2. 预防RNase污染，应注意以下几方面：

1）使用无RNase的塑料制品和枪头，避免交叉污染。

2）玻璃器皿应在使用前于180℃高温下干烤4小时，塑料器皿可在0.5 M NaOH中浸泡10分钟，用水彻底冲洗后高压灭菌。

3）配制溶液应使用无RNase的水。

4）操作人员戴一次性口罩和手套，实验过程中要勤换手套。

3. 提取的样品避免反复冻融，否则影响RNA提取的量和质量。

4. Buffer RL在使用前请加入β-巯基乙醇，1 ml Buffer RL加10 μl β-巯基乙醇。加入β-巯基乙醇的Buffer RL室温可保存1个月。Buffer RLC使用时不需加β-巯基乙醇。

5. 第一次使用前应按照试剂瓶标签的说明先在Buffer RW2中加入无水乙醇。

6. Buffer RL和Buffer RLC如果产生沉淀，请加热使其溶解后室温放置。

7.所有离心步骤均在室温下进行，且所有操作步骤动作要迅速。

操作步骤

1．50-100 mg植物组织在液氮中迅速研磨成粉末，加入600 μl Buffer RL（使用前检查是否加入β-巯基乙醇）或Buffer RLC。涡旋振荡使其充分裂解。

注意：1）Buffer RL主要成分为异硫氰酸胍，适用于大多数植物组织的裂解。但有些植物组织（如玉米的胚乳），由于次级代谢产物较特殊，异硫氰酸胍使样品产生沉淀，导致RNA提取效果不佳，此时可加入Buffer RLC替代Buffer RL。

2）56℃孵育1-3分钟有助于组织的裂解，但是淀粉含量高的植物不要进行高温孵育。

2. 将步骤1所得全部液体转移至已装入收集管的吸附柱(Spin Columns FL)中，12,000 rpm（~13,400×g）离心2分钟，将收集管中的上清液转移到一个新的离心管（自备）中。

注意：1）在吸取液体时可以将枪头尖端剪掉，便于取样。

2）Spin Columns FL可以除去大部分的碎片，但仍会有小部分流出，离心后会在收集管内形成沉淀，在进行下一步时注意避免吸到沉淀。

3. 在步骤2所得干净的裂解液中加入0.5倍体积的无水乙醇，迅速混匀。

注意：加入乙醇后可能会产生沉淀，但不影响后续试验。

4. 将上步得到的溶液转移到已装入收集管的吸附柱（Spin Columns RM）中,若一次不能将全部溶液加入吸附柱中，请分两次转入; 12,000 rpm离心15秒，弃掉废液，将吸附柱重新放回收集管中。

5. 向吸附柱中加入350 μl Buffer RW1，12,000 rpm离心1分钟，弃废液，将吸附柱重新放回收集管中。

6. 配制DNase I 混合液：取52 μl RNase-Free Water，向其中加入8 μl 10×Reaction Buffer和20 μl DNase I（1 U/μl），混匀，配制成终体积为80 μl的反应液。

7. 向吸附柱中直接加入80 µl DNase I 混合液，20-30℃孵育15分钟。

8. 向吸附柱中加入350 μl Buffer RW1, 12,000 rpm离心1分钟，弃废液，将吸附柱重新放回收集管中。

9. 向吸附柱中加入500 μl Buffer RW2（使用前检查是否加入无水乙醇）, 12,000 rpm离心15秒,弃废液。

10. 重复步骤9。

11. 将吸附柱放回收集管中，12,000 rpm离心1分钟，将吸附柱置于室温数分钟，以彻底晾干吸附柱中的无水乙醇。

注意：这一步的目的是将吸附柱中残余乙醇去除，乙醇残留会影响后续的酶促反应(酶切、PCR等)。

12. 将吸附柱装入新的离心管中，向吸附膜的中间加入30-50 μl RNase-Free Water，室温放置1分钟，12,000 rpm离心1分钟，得到的RNA溶液保存在-70℃，防止降解。注意：1） RNase-Free Water体积不应小于30 μl，体积过小影响回收率。

2） 如果要提高RNA的产量，可用30-50 μl新的RNase-Free Water重复步骤12。

3） 如果要提高RNA浓度，可将得到的溶液重新加入到吸附柱中，重复步骤12。

R669988	Component	50T	Storage
R669988A	DNase I	1000 U	-20℃. Avoid freeze/thaw cycle.
R669988B	10×Reaction Buffer	1000 μL	-20℃. Avoid freeze/thaw cycle.
R669988C	Buffer RL	35 mL	RT
R669988D	Buffer RLC	35 mL	RT
R669988E	Buffer RW1	40 mL	RT
R669988F	Buffer RW2 (concentrate)	11 mL	RT
R669988G	RNase-Free Water	10 mL	RT
R669988H	Spin Columns FL with Collection Tubes	50 sets	RT
R669988I	Spin Columns RM with Collection Tubes	50 sets	RT
R669988J	RNase-Free Centrifuge Tubes (1.5 mL)	50 EA	RT

Products

This kit is used for the extraction and purification of high-quality total RNA from a variety of plants, and is also suitable for the extraction of fungal mycelial RNA. The unique separation column is used for homogenization and filtration of high viscosity plant or fungal lysates, while the silicon matrix membrane is used to adsorb the RNA for purification, so that various contaminants, such as polysaccharides, are effectively removed by washing, and the eluted RNA can be directly used in various downstream experiments. The molecular weight of RNA extracted by this kit is more than 200 bases, with high purity and almost no DNA residue. For RNA experiments that are very sensitive to trace DNA, the residual DNA can be removed by digestion on a column using RNase-free DNase. The extracted RNA can be used in Northern Blot, Dot Blot, RT-PCR and in vitro translation experiments.

Self-contained reagents: β-mercaptoethanol, anhydrous ethanol (freshly opened or for RNA extraction).

Pre-experiment Preparation and Important Notes

1. To prevent RNase contamination, attention should be paid to the following aspects:

1) Use RNase-free plastics and tips to avoid cross-contamination.

2) RNase-free water should be used to prepare the solution.

(3) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.

2. To prevent RNase contamination, attention should be paid to the following aspects:

1) Use RNase-free plastics and tips to avoid cross-contamination.

(2) Glassware should be dry-roasted at 180°C for 4 hours before use, and plasticware can be soaked in 0.5M NaOH for 10 minutes, rinsed thoroughly with water and autoclaved.

3) RNase-free water should be used to prepare the solution.

(4) Operators wear disposable masks and gloves, and change gloves diligently during the experiment.

3. Avoid repeated freezing and thawing of the extracted samples, otherwise it will affect the amount and quality of RNA extraction.

4. Please add β-mercaptoethanol to Buffer RL before use, add 10μl of β-mercaptoethanol to 1ml of Buffer RL, it can be stored for 1 month at room temperature. Buffer RL with β-mercaptoethanol can be stored at room temperature for 1 month. β-mercaptoethanol is not required for use of Buffer RLC.

5. Anhydrous ethanol should be added to Buffer RW2 before first use according to the instructions on the reagent bottle label.

6. If precipitation occurs in Buffer RL and Buffer RLC, heat to dissolve and leave at room temperature.

7. All centrifugation steps are carried out at room temperature and all steps are performed quickly.

Procedure

1. 50-100 mg of plant tissue is quickly ground to a powder in liquid nitrogen and added to 600 μl of Buffer RL (check for addition of β-mercaptoethanol before use) or Buffer RLC. vortexing and oscillating to allow for adequate lysis.

Note: 1) The main component of Buffer RL is guanidine isothiocyanate, which is suitable for lysis of most plant tissues. However, in some plant tissues (e.g. endosperm of corn), due to the special secondary metabolites, guanidine isothiocyanate causes precipitation of the sample, resulting in poor RNA extraction, in this case, Buffer RLC can be added instead of Buffer RL.

2) Incubation at 56°C for 1-3 minutes helps tissue lysis, but do not incubate at high temperatures for plants with high starch content.

2. Transfer all the liquid obtained in step 1 to an adsorption column (Spin Columns FL) that has been loaded into a collection tube, centrifuge at 12,000 rpm (~13,400 x g) for 2 minutes, and transfer the supernatant from the collection tube to a new centrifuge tube (supplied).

Note: 1) The tip of the tip of the gun can be cut off when aspirating liquids to facilitate sampling.

2) Spin Columns FL removes most of the debris, but a small portion will still flow out and a precipitate will form in the collection tube after centrifugation, so be careful to avoid aspirating the precipitate when proceeding to the next step.

3. Add 0.5 times the volume of anhydrous ethanol to the clean lysate obtained in step 2 and mix rapidly.

Note: Precipitation may occur upon addition of ethanol, but does not affect subsequent tests.

4. Transfer the solution obtained in the previous step to the Spin Columns RM in the collection tube. If it is not possible to add all of the solution to the column at one time, centrifuge the column at 12,000 rpm for 15 seconds in two batches, discard the waste solution and put the column back into the collection tube.

5. Add 350 μl Buffer RW1 to the adsorbent column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid and put the adsorbent column back into the collection tube.

6. Preparation of DNase I mixture: Take 52μl of RNase-Free Water, add 8μl of 10×Reaction Buffer and 20μl of DNase I (1U/μl) to it, mix well, and make a final volume of 80μl of reaction solution.

7. Add 80µl of DNase I mixture directly to the adsorption column and incubate at 20-30°C for 15 minutes.

8. Add 350 μl of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the waste liquid and put the column back into the collection tube.

9. Add 500 μl of Buffer RW2 to the column (check that anhydrous ethanol is added before use), centrifuge at 12,000 rpm for 15 seconds, and discard the waste solution.

10. Repeat step 9.

11. Place the adsorbent column back into the collection tube, centrifuge at 12,000 rpm for 1 minute, and allow the column to come to room temperature for a few minutes to thoroughly dry out the anhydrous ethanol in the adsorbent column.

Note: The purpose of this step is to remove residual ethanol from the adsorption column; ethanol residue can interfere with subsequent enzymatic reactions (zymography, PCR, etc.).

12. Load the adsorption column into a new centrifuge tube, add 30-50 μl of RNase-Free Water to the middle of the adsorbent membrane, leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 1 minute, and store the resulting RNA solution at -70°C to prevent degradation.

Note: 1) The volume of RNase-Free Water should not be less than 30 μl, too small volume affects the recovery rate.

2) If you want to increase the RNA yield, repeat step 12 with 30-50 μl of fresh RNase-Free Water.

3) If the RNA concentration is to be increased, the resulting solution can be reintroduced into the adsorption column and step 12 repeated.

AI解读

安全和危险性（GHS）

SDS英文版

技术规格说明书

技术规格说明书

r669988_rnapure plant kit（dnase i）.pdf

质检证书（CoA,COO,BSE/TSE 和分析图谱)

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

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复溶计算器

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植物RNA提取试剂盒（DNase I）

库存信息

基本描述

AI解读

安全和危险性（GHS）

技术规格说明书

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