Highlights
- Easy Handling: Bypass chloroform, phase separation and precipitation steps.
- NGS-Ready: Ultra-pure RNA without phenol carryover. No DNA contamination (DNase I included).
- Non-Biased: Complete RNA recovery without miRNA loss.
Description
| Compatibility | TRIzol®, RNAzol®, QIAzol®, TriPure™, TriSure™ and all other acid-guanidinium-phenol based solutions can be used in place of TRI Reagent®. |
|---|---|
| Equipment | Microcentrifuge, vortex |
| Sample Inactivation | TRI Reagent® (provided with R2061, R2063) inhibits RNase activity and inactivates viruses and other infectious agents. |
| Sample Source | Any sample stored and preserved in TRI Reagent®, TRIzol® or similar (animal cells, tissue, bacteria, yeast, fecal, biological fluids, and in vitro processed RNA (e.g., transcription products, DNase-treated or labeled RNA)). |
| Size Range | Total RNA ≥ 17 nt |
| Yield | 10 µg RNA (binding capacity), ≥6 µl (elution volume) |
Q1: Is Direct-zol suitable for very small numbers of cells?
Yes, the Direct-zol MicroPrep (#R2060) is designed and capable of purifying RNA down to single cell inputs (picogram amounts). A sensitive quantification method is needed (e.g. Qubit, qPCR, etc.)
Q2: Is DNase I available for individual purchase?
All kit components are available for purchase separately.
Q3: How to store DNase-I following resuspension?
Lyophilized DNase I is stable at room temperature. Once resuspended, store frozen aliquots. Minimize freeze thaw cycles as much as possible. Freeze thaw will lower DNase activity.
Q4: Is the DNase-I treatment necessary?
If the downstream application requires DNA-free RNA, we recommend performing the DNase I treatment.
Q5: Is the kit compatible with samples stored in DNA/RNA Shield?
Yes, bring samples homogenized and stored in DNA/RNA Shield to room temperature (20-30ºC). Add 3 volume of TRIzol/TRI Reagent and mix well. Proceed with RNA Purification.
Q6: Is it possible to extract proteins with the Direct-zol RNA kits?
Yes, proteins can be Acetone Precipitated post RNA binding step. Please request supplementary protocol from Zymo Research Technical Support.
Q7: I ran out of RNA Wash Buffer. Can I use something else?
Yes, use 80% ethanol as a substitute. RNA Wash Buffer is also sold separately.
Q8: Can samples be stored in TRIzol/TRI Reagent prior to processing?
Yes, samples in TRIzol/TRI Reagent or similar are stable overnight at room temperature and can be stored frozen (-80C). Be sure to lyse and homogenize the sample well prior to freezing. Bring the sample to room temperature prior to RNA Purification.
Q9: Is it possible to isolate DNA with the Direct-zol RNA kits?
Direct-zol DNA/RNA (D2080) kits can isolate DNA from TRIzol
Q10: Is the RNA suitable for Next-Gen sequencing or other sensitive downstream applications?
Yes, the RNA is high quality (A260/A280 >1.8, A260/A230 >1.8) and suitable for any downstream application, including NGS, RT-PCR, hybridization, etc.
Q11: Which phenol-based reagents are compatible with Direct-zol?
The Direct-zol kits are compatible with TRI Reagent, TRIzol, Qiazol, RNAzol, TriPure, TriSure, etc., and any other acid-guanidinium phenol-based reagents.
Q12: What is the difference between the Direct-zol RNA and Quick-RNA kits?
Direct-zol is for samples stored/collected into TRIzol/similar reagents. Quick-RNA is for all other samples.
Q13: What is the difference between the Direct-zol RNA MiniPrep and the Direct-zol RNA MiniPrep Plus?
Both kits function the same, the only difference is the RNA binding capacity of the column provided with the kit.
“Before I discovered this kit, I was isolating RNA the old school way with chloroform and it would take half the day to finish the protocol. The Direct-zol RNA Miniprep kit is AWESOME!It took hardly any time, the protocol was so easy, and my RNA quality was SO much better. Honestly, this kit revolutionized my life at the bench.”
-A. Newhart (The Wistar Institute)
“Simple protocol and yielded good quality of RNA. Only one kit working for all type of tissue, cell and especially biological fluids.”
-Mohan K. (University of Illinois, Chicago)
“Previously I used a protocol that took 3 hours, now I can have my RNA in 20 minutes. What is not to like about that? Just one column and two buffers, I love it.”
-Arjan V. (Indiana University)
Read More| Cat # | Name | Size | Price | |
|---|---|---|---|---|
| E1010-1-16 | DNA Digestion Buffer | 16 mL | $29.00 | |
| R2050-1-50 | TRI Reagent | 50 ml | $70.00 | |
| R2050-1-200 | TRI Reagent | 200 ml | $219.00 | |
| E1010-1-4 | DNA Digestion Buffer | 4 mL | $15.00 | |
| W1001-10 | DNase/RNase-Free Water | 10 ml | $19.00 | |
| W1001-4 | DNase/RNase-Free Water | 4 ml | $12.00 | |
| R2050-2-160 | Direct-zol RNA PreWash (Concentrate) | 160 ml | $166.00 | |
| R2050-2-40 | Direct-zol RNA PreWash (Concentrate) | 40 ml | $42.00 | |
| C1001-50 | Collection Tubes | 50 Pack | $15.00 | |
| R2062 | Direct-zol RNA Microprep | 200 preps | $545.00 | |
| R2060 | Direct-zol RNA Microprep | 50 preps | $175.00 | |
| C1004-50 | Zymo-Spin IC Columns | 50 Pack | $53.00 | |
| R1003-3-48 | RNA Wash Buffer | 48 ml | $105.00 | |
| R1003-3-12 | RNA Wash Buffer | 12 ml | $30.00 | |
| E1010 | DNase I Set | 250 U | $56.00 |
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既说像坐标画曲线曲线应纵轴该荧光强度吧轴数值变化代表荧光强度变化荧光强度变化代表钙离浓度变化 我觉直接用荧光强度值变化代替钙离浓度变化研究意义该钙离浓度变化吧
非要荧光强度值换算钙离浓度需要做列工作
1 取标准钙离浓度染色用共聚焦显微镜测荧光强度
2 标准品需要同像环境主要曝光间相同激光波及能量
3 标准品要用同浓度荧光染料
才能根据标准荧光强度关系推算品钙离浓度
这些染料的结构式基本是保密的。有些地方能查到,象维基上就有SYBR Green的结果式,但谁也不能肯定是否是正确的。
从大肠杆菌细胞中分离质粒DNA的方法众多，其分离的依据可利用分子大小不同，碱基组成的差异以及质粒DNA的超螺旋共价闭合环状结构的特点来进行。碱基性法抽提效果良好，既经济且得率较高。抽提到的质量DNA可用于酶切、连接和转化。对于分子量较大拷贝较少对的质粒DNA，由于DNA片段较大易于损伤断裂，因此选用吕华铯密度超高离心法抽提DNA，且具有纯度高、步骤少、方法稳定且获得的质量DNA是超螺旋构型等特点。对于高拷贝数质粒，用少量制备法抽提质粒DNA就有足够量可用于基因操作。
质粒DNA纯化的试验步骤：
测量DNA溶液的体积，按lg/ml的用量精确地加入固体CsCl, 将溶液加温至30℃助溶。温和地混匀溶液直到盐溶解。
每10mlDNA溶液加入0.8ml溴化乙锭溶液（10mg/ml溶于水）；立即将溴化乙锭溶液（漂浮在表层）与DNA－氯化铯溶液混匀， 溶液的终密度应为1.55g/ml（溶液的折射率为1.3860）溴化乙锭浓度大约740μg/ml。【溴化乙锭贮存液应贮存于避光容器内（如用锡箔完全包裹的瓶子），于室温保存。
室温下用Sorvall SS34头（或与其相当的转头）以8000rpm离心5min， 浮在溶液上面的水垢状浮渣是溴化乙锭和细菌蛋白所形成的复合物。
用巴斯德吸管或带大号针头的一次性注射器将浮渣下的清亮红色溶液转移到离心管中。用轻石蜡油加满管的其余部分并封口。
以20℃对所得的密度梯度以45 000rpm离心16h（VTi65 转头）、 以45 000rpm离心48h（Ti50转头）、以60 000rpm离心24h（Ti65转头）或者以60 000rpm离心24h（Ti70.1转头）。普通光照下，在梯中心可见两条DNA区带， 上部区带材料通常较少，由线状的细菌（染色体）DNA和带切口的环状质粒DNA组成：下部区带则由闭环质粒DNA组成。管底部深红色的沉淀是溴化乙锭RNA复合物，位于CsCl溶液和石蜡油之间的是蛋白质。Beckman Quick-Seal离心管中的CsCl－溴化乙锭梯度可容纳4mg 闭环质粒DNA而不至超负荷。如有更大量的质粒存在，将扩展为一条宽带，并与染色体DNA相重叠。这种问题只有在质粒复制达到极高水平时才会出现，只要将该质粒提取物分为2个梯度即可解决。如出现负荷，可收集整个DNA区高产水平时才会出现，只要将该质粒提取物分为2个梯度即可解决。如出现超负荷，可收集整个DNA区带，用CsCl溶液（ρ=1.58g/ml）将体积调到15ml，在两个离心管中再度离心，使DNA达到平衡。
收集DNA带。将21 号皮下注射针头插入管的顶端以使空气进入，为尽量减少污染的机会，首先用18号皮下注射针头按下述方法收集上部的区带（杂色体DNA）：用乙醇小心擦拭管外壁以除去任何油脂，然后将一块Soctch胶带贴于管外壁。穿过Soctch胶带将18号皮下注身针头（其斜面向上）插入管中，以便使针头的斜面开口恰好位于染色体DNA区带之下并与该区带相平行。将粘稠状DNA收集到一次性使用的管内，用造型粘土块封住皮下注射针头的末端并将第2根针头留于原处。 穿过Soctch 胶带插入第3根皮下注射针头（18号），将下部的质粒DNA区带收集到玻璃或塑料管中。展开
什么是纯荧光校正，多长时间校正一次：
纯荧光校正是测定各种纯荧光染料标准品的波长和信号强度，通俗地说是让仪器“认识”各种荧光染料。软件收集并储存各种纯荧光染料标准品的荧光信息。以后每次定量实验运行过程中，SDS软件收集样品的原始光谱信号，并将此原始光谱与纯荧光文件中的数据进行比较，精确扣除不同染料的信号重叠部分，从而确定样品中的荧光染料种类和信号强度。
推荐每半年进行一次纯荧光校正。在运行光谱校正之前，请先进行背景校正和ROI校正。
