本试剂盒采用了经过优化的缓冲液系统，适合从细菌样本中提取总RNA。使用本试剂盒可以有效提取分子量大于200nt的RNA。
本试剂盒具有高效、快速和方便的特点，提取过程中也无需苯酚氯仿抽提等步骤。利用该试剂盒提取的RNA纯度高，极少含蛋白质、基因组DNA和其他杂质的污染。提取得到的RNA可以直接用于Northern杂交、斑点杂交、mRNA纯化、 体外翻译、RNA分解酶的保护分析、RT-PCR、RT-qPCR和构建cDNA文库等各种分子生物学实验。
储存条件
R669890A, R669890B, R669890C, R669890D, R669890E, R669890F, R669890G, R669890H, R669890I：室温储存，可保存15个月。
R669890J, R669890K, R669890L: 2-8℃储存，可保存15个月。
加入β-巯基乙醇的Buffer URL 4℃可保存1个月。
注意事项
1. 操作前在Buffer URL中加入β-巯基乙醇至终浓度1%，配好的URL可在4℃放置1个月。Buffer URL在储存时可能会形成沉淀，如果有沉淀出现，请于37℃加热溶解，然后待恢复至室温后使用。
2. 尽可能使用新鲜收集的样品，RNA产量与起始样品中RNA的完整性有关，按照说明书标准流程降解为小片段的RNA不能被有效回收。
3. DNase I储存液配制：用1 mL注射器将550 μL RNase-Free ddH₂O注入装有DNase I干粉的瓶中，轻柔混匀至溶解，分装后-30 ~ -15℃储存（可保存9个月），每次根据用量需求取出。配制的 DNase I工作液置于2-8℃储存（可保存42天），避免反复冻融。
操作步骤
使用前请先在12 mL的Buffer RW2中加入48 mL无水乙醇。
1. 4℃ 12,000rpm (13,400× g) 离心2 min收集菌体（收集菌体的最大量不超过1×10⁹），小心去除所有培养基上清，后续的离心步骤均在室温进行。
2. 用100 μL含有溶菌酶的Buffer TE彻底重悬菌体，37℃孵育。溶菌酶使用量及孵育时间见下表。
细菌种类
| Buffer TE中溶菌酶终浓度
| 孵育时间（37℃）
|
G-细菌
| 500 μg/mL
| 3-5 min
|
G+细菌
| 3 mg/mL
| 5-10 min
|
TE缓冲液配方：50mM Tris, pH 8.0；10mM Na₂-EDTA，pH 8.0。121℃灭菌处理20 min。
例：
G-细菌：1 μL 溶菌酶储存液 (50 mg/mL) +99 μL Buffer TE，重悬菌体，溶菌酶终浓度即为500 μg/mL；
G+细菌：6 μL 溶菌酶储存液 (50 mg/mL) +94 μL Buffer TE，重悬菌体，溶菌酶终浓度即为3 mg/mL。
3. 加入350 μL Buffer URL（使用前请先加入β-巯基乙醇），涡旋振荡混匀15 sec（此步骤可能出 现沉淀）。将溶液与沉淀全部加入到过滤柱GS（过滤柱放在收集管中），12,000 rpm (13,400 × g) 离心1 min。
4. 将滤液转移至一个新的1.5 mL RNase-Free离心管中（不要吸取到底部沉淀），加入250 μL无水乙醇，混匀（此时可能会出现沉淀），将得到的溶液和沉淀一起转入吸附柱RA（吸附柱放在收集管中) ，12,000 rpm (13,400 × g) 离心30 sec，弃废液，将吸附柱放回收集管中。
5. 向吸附柱中加入350 μL Buffer RW，12,000rpm (13,400×g) 离心30 sec，弃废液，将吸附柱放回收集管中。
6. DNase I工作液的配制：取10 μL DNase I储存液放入新的RNase-Free离心管中，加入70 μL DNase I Buffer，轻柔混匀。
7. 向吸附柱中央加入80 μL的DNase I工作液，室温放置15 min。
8. 向吸附柱中加入350 μL Buffer RW，12,000rpm (13,400×g)离心30 sec，弃废液，将吸附柱放回收集管中。
9. 向吸附柱中加入500 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm (13,400 × g) 离 心30 sec，弃废液，将吸附柱放回收集管中。
10. 重复步骤9。
11. 12,000rpm (13,400 × g) 离心2 min。
12. 将吸附柱转移至一个新的1.5 mL RNase-Free离心管中，打开吸附柱管盖，室温放置1 min，以彻底晾干吸附柱中的无水乙醇。向吸附膜的中间部位悬空滴加30-100 μL RNase-Free ddH₂O，12,000 rpm (13,400 × g) 离心30 sec。得到的RNA可直接用于下游实验，或于-80℃以下保存。
注意：RNase-Free ddH₂O体积不应小于30 μL，体积过小影响回收率。若要提高RNA的产量，可将第一次洗脱液重新加入吸附柱进行洗脱，或将RNase-Free ddH₂O置于65℃预热。
This kit utilizes an optimized buffer system, specifically designed for extracting total RNA from bacterial samples. It can effectively isolate RNA molecules with a molecular weight greater than 200 nt.
The kit features high efficiency, rapid processing, and ease of use, eliminating the need for phenol-chloroform extraction steps. RNA purified with this kit exhibits high purity, with minimal contamination from proteins, genomic DNA, and other impurities. The isolated RNA can be directly used in various molecular biology experiments, including Northern blotting, dot blotting, mRNA purification, in vitro translation, RNase protection assays, RT-PCR, RT-qPCR, and cDNA library construction.
Storage Conditions
- R669890A, R669890B, R669890C, R669890D, R669890E, R669890F, R669890G, R669890H, R669890I: Stable for 15 months when stored at room temperature.
- R669890J, R669890K, R669890L: Stable for 15 months when stored at 2-8°C.
- Buffer URL supplemented with β-mercaptoethanol can be stored at 4°C for 1 month.
Precautions
1. Buffer URL Preparation: Before use, add β-mercaptoethanol to Buffer URL to a final concentration of 1%. The prepared Buffer URL can be stored at 4°C for 1 month. Precipitates may form in Buffer URL during storage; if precipitates occur, heat the solution at 37°C to dissolve, then allow it to cool to room temperature before use.
2. Sample Freshness: Use freshly harvested bacterial samples whenever possible. RNA yield depends on the integrity of RNA in the starting material; RNA degraded into small fragments during sample processing cannot be effectively recovered.
3. DNase I Stock Solution Preparation: Inject 550 μL of RNase-Free ddH₂O (from R669890L) into the vial containing DNase I powder using a 1 mL syringe, mix gently until dissolved. Aliquot the solution and store at -30 ~ -15°C (stable for 9 months). Prepare working DNase I solution as needed and store at 2-8°C (stable for 42 days). Avoid repeated freeze-thaw cycles.
Protocol
Preparatory Step: Add 48 mL of anhydrous ethanol to 12 mL of Buffer RW2 before use.
1. Harvest Bacterial Cells: Centrifuge the bacterial culture at 12,000 rpm (13,400 × g) for 2 min at 4°C to harvest cells (maximum cell number ≤ 1×10⁹). Carefully remove all supernatant. Subsequent centrifugation steps should be performed at room temperature.
2. Resuspend and Lyse Cells: Resuspend the cell pellet thoroughly in 100 μL of Buffer TE containing lysozyme. Incubate at 37°C. Refer to the table below for lysozyme concentration and incubation time:
Bacteria Type
| Final Lysozyme Concentration in Buffer TE
| Incubation Time at 37°C
|
Gram-negative (G⁻)
| 500 μg/mL
| 3-5 min
|
Gram-positive (G⁺)
| 3 mg/mL
| 5-10 min
|
TE Buffer Formula: 50 mM Tris (pH 8.0), 10 mM Na₂-EDTA (pH 8.0). Autoclave at 121°C for 20 min.
Example:
- For G⁻ bacteria: Mix 1 μL lysozyme stock solution (50 mg/mL) with 99 μL Buffer TE to achieve a final concentration of 500 μg/mL.
- For G⁺ bacteria: Mix 6 μL lysozyme stock solution (50 mg/mL) with 94 μL Buffer TE to achieve a final concentration of 3 mg/mL.
3. Lyse Cells: Add 350 μL of Buffer URL (supplemented with β-mercaptoethanol before use), vortex vigorously for 15 sec (precipitates may form). Transfer the entire mixture (including precipitates) to Filter Column GS (placed in a collection tube), centrifuge at 12,000 rpm (13,400 × g) for 1 min.
4. Bind RNA to Adsorption Column: Transfer the filtrate to a new 1.5 mL RNase-Free centrifuge tube (do not aspirate the bottom precipitate). Add 250 μL of anhydrous ethanol and mix well (precipitates may form). Transfer the entire mixture to Adsorption Column RA (placed in a collection tube), centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
5. Wash Column: Add 350 μL of Buffer RW to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
6. Prepare DNase I Working Solution: Combine 10 μL of DNase I stock solution with 70 μL of DNase I Buffer in a new RNase-Free centrifuge tube, mix gently.
7. Remove Genomic DNA: Add 80 μL of DNase I working solution to the center of the adsorption column membrane. Incubate at room temperature for 15 min.
8. Post-DNase Wash: Add 350 μL of Buffer RW to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
9. Final Wash: Add 500 μL of Buffer RW2 (supplemented with anhydrous ethanol before use) to the column, centrifuge at 12,000 rpm (13,400 × g) for 30 sec. Discard the flow-through and place the column back into the collection tube.
10. Repeat Final Wash: Repeat step 9.
11. Dry Column: Centrifuge the column at 12,000 rpm (13,400 × g) for 2 min to remove residual ethanol.
12. Elute RNA: Transfer the adsorption column to a new 1.5 mL RNase-Free centrifuge tube. Open the column cap and air-dry for 1 min at room temperature to completely remove residual ethanol. Add 30-100 μL of RNase-Free ddH₂O dropwise to the center of the membrane, then centrifuge at 12,000 rpm (13,400 × g) for 30 sec. The eluted RNA can be used directly in downstream experiments or stored at ≤ -80°C.
Note: The volume of RNase-Free ddH₂O should not be less than 30 μL, as smaller volumes will reduce recovery. To increase RNA yield, re-apply the first eluate to the column for a second elution, or pre-heat the RNase-Free ddH₂O to 65°C before elution.
G2629163
危险品化学品经营许可证（带存储）