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®. |
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Equipment | Microcentrifuge, vortex |
Sample Inactivation | TRI Reagent® (provided with R2051, R2053) 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 | 50 µg RNA (binding capacity), ≥25 µl (elution volume) |
Q1: Is DNase I available for individual purchase?
All kit components are available for purchase separately.
Q2: 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.
Q3: 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.
Q4: 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.)
Q5: 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.
Q6: Is the DNase-I treatment necessary?
If the downstream application requires DNA-free RNA, we recommend performing the DNase I treatment.
Q7: 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.
Q8: Is it possible to isolate DNA with the Direct-zol RNA kits?
Direct-zol DNA/RNA (D2080) kits can isolate DNA from TRIzol
Q9: 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.
Q10: 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.
Q11: 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.
Q12: 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.
Q13: 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.
“No phase separation was needed, but you still had the benefits of a Trizol extraction. No need to precipitate and resuspend samples, which means less sample loss during purification.”
-Adina B. (University of Guelph)
“This kit is amazing, I"ve got a gel comparing the lack of gDNA as shown in the advertising pamphlet. What can I say, except: I love this product!“
-R.K. CSU
“Direct-zol is the most excellent kit for RNA isolation that I ever used in the past 20 years.”
-H.Z. (Harvard Medical School)
Read MoreCat # | Name | Size | Price | |
---|---|---|---|---|
E1010-1-4 | DNA Digestion Buffer | 4 mL | $15.00 | |
E1010-1-16 | DNA Digestion Buffer | 16 mL | $29.00 | |
R2050-1-200 | TRI Reagent | 200 ml | $219.00 | |
R2050-1-50 | TRI Reagent | 50 ml | $70.00 | |
C1078-50 | Zymo-Spin IICR Columns | 50 Pack | $55.00 | |
R1003-3-12 | RNA Wash Buffer | 12 ml | $30.00 | |
R1003-3-48 | RNA Wash Buffer | 48 ml | $105.00 | |
E1010 | DNase I Set | 250 U | $56.00 | |
R2050-2-40 | Direct-zol RNA PreWash (Concentrate) | 40 ml | $42.00 | |
R2050-2-160 | Direct-zol RNA PreWash (Concentrate) | 160 ml | $166.00 | |
C1001-50 | Collection Tubes | 50 Pack | $15.00 | |
R2050 | Direct-zol RNA Miniprep | 50 preps | $180.00 | |
R2052 | Direct-zol RNA Miniprep | 200 preps | $555.00 | |
W1001-6 | DNase/RNase-Free Water | 6 ml | $15.00 | |
W1001-30 | DNase/RNase-Free Water | 30 ml | $22.00 |
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新手一枚,看到论坛里也有许多人问过,但没找到比较确切的回答,还是不是很理解,做芯片给出的lncRNA的名字是XLOC_009167,这个名字在ncbi里面都找不出来,我该怎么样去获得这个rna的信息呢?
引物是一小段单链DNA或RNA,引物可以做为DNA复制开始时DNA聚合酶的结合位点,在细胞外的条件下,只有通过引物,DNA才可以开始进行复制。
引物是人工合成的两段寡核苷酸序列,一个引物与感兴趣区域一端的一条DNA模板链互补,另一个引物与感兴趣区域另一端的另一条DNA模板链互补。
启动子和引物两者的区别:
启动子是DNA分子上能与RNA聚合酶结合并形成转录起始复合体的区域,在许多情况下,还包括促进这一过程的调节蛋白的结合位点,决定RNA聚合酶转录起始位点的DNA序列。引物是一小段单链DNA或RNA,引物可以做为DNA复制开始时DNA聚合酶的结合位点,在细胞外的条件下,只有通过引物,DNA才可以开始进行复制。引物是人工合成的两段寡核苷酸序列,一个引物与感兴趣区域一端的一条DNA模板链互补,另一个引物与感兴趣区域另一端的另一条DNA模板链互补。
启动子是位于结构基因前的非编码区对转录起调控作用的一段DNA序列。引物则是游离于DNA复制的模板链之外的对DNA复制起调控作用的一小段单链DNA或RNA。
需要在片段5‘端和3’端分别设计引物去扩增。
所以,对应的上游5‘端,我们习惯叫做”上游引物“,即forward primer,简称for;
对应序列的3’端,我们叫做下游引物,reverse primer,简称rev。
我们在设计pcr引物的时候,经常需要将设计好的正反向引物与基因组DNA序列进行比对验证,但是因为反向引物是反向互补的,所以有些麻烦。因此,在这里介绍两个非常的小工具,一个是在线的模拟PCR,一个是本地运行的程序包,我会以附件形式上传,不需要蚁豆即可下载。两个都写得非常详细,相信大家都能看懂。不知道这个帖子能不能加分?希望版主看到后给予加分奖励
方法一:UCSC中的In-SilicoPCR
1、登录UCSC,网址链接http://www.genome.ucsc.edu/index.html,点击右侧工具的In-SilicoPCR,或者在Tools下拉框中点击In-SilicoPCR,如下所示:
2、点击In-SilicoPCR后,出现如下界面,我们需要将自己设计好的正向引物和反向引物分别粘贴至各自空白框,点击submit。对其它几个参数做点说明吧,MaxProductSize是指被放大区域的最大长度,MinPerfectMatch是指和引物的3‘末端PerfectMatch的最小碱基数目,MinGoodMatch:是指和引物的3‘末端GoodMatch的最小碱基数目,GoodMatch是3个碱基里最少匹配2个,FlipReversePrimer:反向引物反向互补。
3、我用自己设计的基因举例说明:HNF-1α的EXON2,参考序列:NM_000545,正向引物5’-3‘:CAGCCCTTGCTGAGCAGATCC,反向引物5’-3‘:CCACTGACTTCCTTTCCATCTACC。正向引物和反向引物分别粘贴至各自空白框,点击submit。出现如下结果,其中第一个红色标记指染色体位置,第二个红色标记指长度,第三个红色标记是我们输入的正反向引物,第四个标记可以直接链接至Primer3在线设计引物,网址http://primer3.ut.ee/。比对染色体位置就可以知道我们设计的引物是否包括HNF-1α的EXON2。
方法二:Reverse程序
这个程序是一位程序员朋友专门为我们编写的,操作非常简单,输入反向互补的引物序列后,就可以得到原始碱基序列。文件夹内一共才3个文件。
举例说明,HNF-1α的EXON2,参考序列:NM_000545,正向引物5’-3‘:CAGCCCTTGCTGAGCAGATCC,反向引物5’-3‘:CCACTGACTTCCTTTCCATCTACC。
1、点击target,粘贴反向引物序列。
2、点击右上角关闭键,这时候会弹出对话框,点击保存。
3、打开文件夹中的reverse_complement应用程度,按任意键即可自动关闭。
4、打开文件夹中的result,就可以得到反向互补引物序列对应的原始序列。
5、这时候就可以将正向序列和刚刚得到的原始序列一起代入HNF-1α的EXON2中检验我们设计的引物是否覆盖了目标片段。
好了,就这两个小工具了,非常实用,希望大家有所收获。有什么疑问可以给我留言或者私信我,支持我的战友就请给我投票吧。版主能给予我加分奖励吗?
reverse_complement.rar(95.82k)
R: Reversed primer 反向引物
在这里我向大家隆重推荐一个在线的引物设计软件,是斯坦福大学的,我认为是最好的在线引物设计软件,我用它设计接近100多对引物,可以说是屡试不爽。它用起来也很简单,最重要的是效果非常好,考虑的因素也非常的多。
先不说那么多了:网址:http://www.yeastgenome.org/cgi-bin/web-primer
下面我继续图示。
帖子发表之后很多战友PM我,让我帮设计引物,其实这很让我失望的,我的本意是让大家掌握这种方法,而不是告诉大家我是一个专家,其实我水平也有限,所以我不会帮大家设计引物,我认为这是科研工作的一部分、基本功,祝大家好运、实验顺利。
accctcccgccccccccgtat
(互补碱基不写了)
那么一般来说,你就要导入以accctc结尾的引物使得增幅继续下去,具体方法和注意事项楼主可以看gee_an大侠的资料.
查了一下测序公司的网页,如果要同定微生物,楼主需要准备如下东东:
1、待测菌体.试管或培养皿都可以,但必须是纯粹的待测菌体,不能含有其他的微生物.
2、提纯的DNA.如果1有致病性或传染性,那么楼主就得自己提纯DNA再交给测序公司.DNA纯度要求可以做PCR.