h**********r 发帖数: 671 | 1 以前做Neurospora的knockout的时候,看到过yeast方面的protocol。感觉他们挺严谨
的。但我不保证work。摘自:
http://www.dartmouth.edu/~neurosporagenome/Projects_files/Proje
Prepare yeast DNA with the Gentra yeast DNA kit:
(the following is slightly modified from their protocol:
http://www.gentra.com/pdf/01160.pdf)
the solutions are available from Gentra as Puregene kit D-6000A or
separately; if you have their Puregene solutions for Neurospora DNA preps,
you only need to order the Cell Suspension Solution (D-6001) and Lytic
Enzyme Sol... 阅读全帖 |
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h**********r 发帖数: 671 | 2 我以前在国内时候提细菌基因组用的方法,貌似对酵母也work,我以前就提过一次酵母
,确实work的很好,还记得gel上的照片贼亮贼亮的。那时候年轻啊!问过别人,说
lysozyme对yeast也能work.
Extraction of DNA from Bacteria and Yeasts
(This protocol was developed by Housepainter on February 09, 2010)
1.Inoculate 5 ml of medium with a single colony of transformed bacteria or
yeasts.
2.Pour 1.0 ml of the culture into a tube. Centrifuge at maximum speed for 30
seconds at room temperature. Remove the supernatant.
3.Resuspend the bacterial pellet in 1.0 ml 0.85% NaCl by vigorous vortexi... 阅读全帖 |
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C*******e 发帖数: 4348 | 3 顶这个
用bioanalyzer的pico chip
然后同时跑RNase-free DNase treated样品做对照
nanodrop低于10ng/ul就不太准了
不过在测核酸方面省样品,实在是个好东西
这年头我以为已经是标配了呢 |
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m*********n 发帖数: 215 | 4 另外分离组织的过程一定注意所有的用具RNase free。除了如果要酶解dissociate
cells,尽量保持放在冰上/低温操作。 |
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b*********b 发帖数: 64 | 5 I use QIAGEN Plasmid Midi kit. 100ml culture could give about 30-50ug. I
tried Qiagen's large construct kit.But in my hand, the midi kit is much much
better than the large construct kit, and also takes much less time.The
following is the protocol I got from Qiagen.
User-Developed Protocol:
Isolation of BAC DNA using the QIAGEN® Plasmid Midi Kit
This procedure has been adapted by customers from the QIAGEN® Plasmid
Midi Kit Protocol.It has not been thoroughly tested and optimized by QIAG... 阅读全帖 |
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m******5 发帖数: 1383 | 6 我觉得你的case应该做deep-sequencing,似乎现在deep sequencing 一个反应也才3千
左右吧,你做半年实验用的光同位素1000就很难打住,加上美国配胶配试剂用的盐也不是
省油的灯,还有RNAse out,还有电费………… 怎么算都比deep sequencing贵
楼主要不把case再说详细点大家一起看看?
gemonez几 |
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A******d 发帖数: 571 | 7 从-80度把肝脏组织拿出来,要量30毫克出来qPCR,怕RNAse,但是在干冰上切割难度很大
,在室温下经常切好了组织差不多化冻了。有没有什么高招? |
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f****n 发帖数: 114 | 8 小弟最近在做RNA in vitro transcription,用的是ambion 的MEGAscript kit。
可是不论是用PCR产物直接做模板,还是连接到质粒上再线性化做模板,都得不到sharp
的条带,size比预期小很多并且有拖尾,但kit里阳性对照可以出来。
已经排除了RNase污染。估计是DNA模板纯度的问题,请问各位PCR产物用啥方法纯化?
另外,我在T7promoter序列前面加了7个碱基,难道是这个影响酶的效率?
请各位高手帮忙! |
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M*****n 发帖数: 16729 | 9 repurify your PCR product and use RNase-free water. |
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g*********d 发帖数: 233 | 10 RNA干扰及其在动物繁殖中的应用
2009-12-28
RNA 干扰(RNA interference, RNAi)是生物进化过程中遗留下来的一种在转录后通
过 RNA 调控基因表达的机制, 它是指在真核细胞中引入双链 RNA分子从而导致具有序
列同源性的基因产生特异性基因沉默 (gene silencing)的现象 。由于 RNA干扰可以阻
断特定的基因表达, 具有超越疫苗和抗病毒药物的诸多优点, 因此在阐述基因功能以及
蛋白质相互作用等方面, 表现出诱人的前景。近年来随着分子生物学的发展, RNAi 的
机制也不断被阐明, 同时国内外研究也证实 RNAi 技术可以为动物繁殖过程中所涉及的
基因功能提供一条新的思路, 这在科研和生产实践上都具有重要的意义。
1 RNAi的发现
Matzke 等于 1989 年报道了启动子介导的序列同源性基因共转染烟草可引起转
基因表达发生沉默。 1990 年, Napoli 和 Van der Krol 等为了让矮牵牛花的颜色更
加鲜艳, 把与紫色相关的查尔酮(chalcone)基因转染到牵牛花内, 结果意外发现, 植物
体的颜色... 阅读全帖 |
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n******2 发帖数: 971 | 11 1、我用的是pcr纯化试剂盒回收dna.
2、0 sonication 的smear有没有可能是RNA?各位在优化sonication的条件时用RNase处
理DNA吗?
3、sonication中,我采用最小功率(大功率极易产生bubble)并依次增加次数的方法
进行优化体系,但始终能在胶上看到有大片段DNA的存在。这是什么原因?sonicate次
数不够(目前已经提高到20多次,15s/pulse)? 还是over cross linking(10min)? |
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z*******a 发帖数: 175 | 12 one more ChIP person, I would avoid ChIP whenever I can.
my comments on your questions.
0 sonication 的smear是RNA, 用RNase处理DNA is necessary
over fixed or sonicator is broken |
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a****o 发帖数: 1786 | 13 1. Optimal crossing has a wide range, usually it does not matter, check
published paper for a reasonable time.
2. You need treat your sample with RNase before running on agarose gel,
otherwise most you see is RNA
3. Promoters are generally depleted of histone and coding regions have high
level of histone. It is not a good choice to use histone. You can detect
histone association even without crossing.
sonicate |
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h****k 发帖数: 182 | 14 使用的是tissue, 书上写的是不要超过30mg, 30mg看起来是个3mm的小方块。过量使用
tissue后效果会怎样?另外加了RLT buffer之后RNase还会降解RNA吗? 书上写的是RLT
buffer含有guanidine thiocyanate. |
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s******y 发帖数: 28562 | 15 关于用量,没有那么严格的。多一点少一点组织无所谓,关键是你得确认所有组织都被
溶掉了,否则就一方面堵树脂,另外一方面带入过多污染物。
你可以多加点裂解液,多处理一回,然后视需要全部倒进一个小管或者分成几个
管。
RNase一般比较皮实,所以没人能保证RLT buffer一定能灭活,该注意的地方还是要注
意的。
RLT |
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n********k 发帖数: 2818 | 16 more buffer and complete and fast lysis will do it...For a beginner, do
yourself a favor and NEVER try to save on buffer---the key is complete
lysis with enough buffer...then it can last fairly long, as long as it is fully lysed, no worry about RNase until the column step...if in doubt, use
RNAlater...it is fantastic...
RLT |
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T**********t 发帖数: 1604 | 18 以前实验室直接用millipore的超纯水,现在的实验室把超纯水再autoclave一下,没啥
问题,用来溶的DNA或者RNA样品放室温几个星期跑出来的胶还是干净的。 |
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M*****n 发帖数: 16729 | 20 this is right.
I believe Millipore water is nuclease free.but make sure you use nuclease-
free container for the water. |
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T**********t 发帖数: 1604 | 21 我个人是认为autoclave超纯水属于多此一举。但是这个新实验室惯例如此。If it
works, don't try to fix it。 |
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j****t 发帖数: 1663 | 23 我是在这个lysis buffer里作sonication的。我现在用0.2% sarkosyl(instead of 0.
5%),和 1% of Na-deoxycholate。sonication的效果很好。而且这个浓度的detegent
对于IP 来说还是高了些,我会在做IP前把sample稀释一倍。
我觉得你的lysis和sonication 的条件也许可以了,问题说不定是在reverse-
crosslink。我的快速检测片段大小和ChIP DNA浓度的步骤如下,供你参考参考。
Check chromatin size and conc.
1. Dilute 50 ul chromatin extracts with 50 ul TE buffer. Add 4 ul of 5 M
NaCl and 1 ul of 10 mg/ml Proteinase K (Invitrogen #25530-015) (use a PCR
tube).
2. Incubate at 65 oC for at lease 4 hr (using a PCR machine) ... 阅读全帖 |
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l**********1 发帖数: 5204 | 24 RE LZ 我就是按你这个做法的,在室温dry了差不多有15-20分钟了
How to put your RNA extraction last process 1.5ml tube for drying?
I mean you put it likes a "U" or "upset U" or "C" or "left direction C"
and on what material? i mean lab tissue paper or not?
RNase free environment including atmosphere You Sure?
plus no proteins contamination You sure too?
htp://www.flychip.org.uk/protocols/gene_expression/rna_qc.pdf
。。 |
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g****e 发帖数: 282 | 25 We are trying to remove all nucleic acids from the supernatant containing
DNA, RNase and cytokines.
DNase and antidote have some side-effects in our system, so it's hard to get
any conclusion.
Is there any way to remove DNA but keep the cytokine activity? |
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r*****t 发帖数: 4793 | 26 and this one
Wiley Interdiscip Rev RNA. 2011 Nov 15. doi: 10.1002/wrna.113. [Epub ahead
of print]
Diversity of animal small RNA pathways and their biological utility.
Okamura K.
Source
Department of Developmental Biology, Sloan-Kettering Institute, New York, NY
, USA. o******[email protected].
Abstract
Higher eukaryotes employ extensive post-transcriptional gene regulation to
accomplish fine control of gene expression. The microRNA (miRNA) family
plays important roles in the post-transcriptional gene ... 阅读全帖 |
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l**********1 发帖数: 5204 | 27 Genomic DNA plus protein (some RNase + enzyme)
contaminated within your Total RNA extraction sample
from gel picture: the most near
414004.jpg
(160922 字节)
that shining line is Genomic DNA absolutely.
for good RNA extraction protocol
please go to
//tools.invitrogen.com/content/sfs/productnotes/F_Trizol%20and%20Trizol%20LS-041018-RD-TL-
HL0506021.pdf |
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M*****e 发帖数: 279 | 29 我的办法可能能帮你解决问题.
我曾经试过多种办法提没有降解(用Bio-Rad or Agilent Bioanalyzer测integrity or
quality (RNA Integrity Number (RIN) by Agilent or RNA Quality Index (RQI)
by Bio-Rad),不是仅仅用Nanodrop测RNA integrity or quality)的total RNA,然后
做microarray.
1.液氮研磨:RNA quality好,可以做microarray,但是麻烦和太慢(原因你应该知道
)。
2. Glass tissue homogenizer: it didn't work for me to grind tissue in
Trizol in ice bucket with ice, although my tissue was thin and tiny.
Bioanalyzer showed RNA degradation.It's also low through-put.
3. Hand-held electr... 阅读全帖 |
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b*******n 发帖数: 8420 | 30 prostate这个组织确实很tricky
因为这玩意本身有很多腺体,在解剖的时候会释放出大量的各种RNase protease之类的
东西,所以只用TRIZOL的话RNA会降解
所以要么就用RNA later,可以保证不降解
要么就是液氮低温处理再加TRIZOL
你最好问问专门做prostate的人要他们的protocol看看,可以少走些弯路 |
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A******y 发帖数: 2041 | 31 Any 1.5mL tubes that is RNAse/DNAse free |
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x**e 发帖数: 163 | 32 我一直用DEPC处理的FAA来固定植物材料,有一次石蜡切片是请别的实验室切的,所有
试剂都没有用DEPC处理,硬着头皮做完了ISH,结果和以前RNAse free的没什么区别。
当然ISH的试剂都是DEPC处理过的。似乎切片过程不太重要,而且材料在石蜡中保存半
年再做ISH也没有问题。 |
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h******y 发帖数: 351 | 33 RNA-RNA has a higher Tm than RNA-DNA, so you can use more stringent washing
in your in situ and get better background. In a nutshell, RNA probe is
better than DNA probe. Of course, your RNA probes should be free of any
RNase contamination.
Current commercially available DIG labeling kits is as sensitive as
radioactive labeling kits, although the advantage of using radioactive-
labeled probes is that you can do quantification analysis by densitometry.
The disadvantage of radioactive-labeled pro... 阅读全帖 |
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c*********r 发帖数: 1312 | 34 formamide强烈抑制RNase,保存在含formamide的溶液里,比如杂交液里,DIG标记的探
针能保存好久好久的。 |
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s******r 发帖数: 2876 | 35 有没有用RNAse later之类的溶液灌注老鼠的,
这样的话RNA会不会保存的好一点,in situ
容易一点。
PF
Isop
做好
子,
Isop |
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s******r 发帖数: 2876 | 36 有没有用RNAse later之类的溶液灌注老鼠的,
这样的话RNA会不会保存的好一点,in situ
容易一点。
PF
Isop
做好
子,
Isop |
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r******g 发帖数: 600 | 37 三级结构不需要二硫键!
DNase I 是没有二硫键的,大家做RNA提纯的时候,不是经常加beta-ME吗? 就是为了
让RNAse失活,再用DNase~ |
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r*****t 发帖数: 4793 | 40 谢谢
这些在细胞里影响转录么? 我想找个只抑制降解,不影响转录(或者影响很小)的东
西 |
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s***o 发帖数: 11 | 41 同意。液氮下研磨后加Tizol提取没问题。
我们存了几千份肿瘤标本,都是离体后马上丢到液氮里,然后转到-80冰箱长期保存。
如果离体后速度降温,中间没有反复升降温度,十年以上都没有问题。
当然有些组织Rnase特别丰富的需要小心些,比如胰腺。 |
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s**u 发帖数: 45 | 42 感觉没sonication开,用RNase处理一下,看下面的带是不是RNA。 |
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C*******e 发帖数: 4348 | 43 既然是试剂盒里自带的control
如果确定没有RNase污染
是没问题的 |
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C*******e 发帖数: 4348 | 44 最近做一个蛋白A
高pI(>9)
已知蛋白A跟蛋白B,或者蛋白B+C有相互作用
于是共表达然后纯化复合物
纯化过程中用detergent啊,lyzosome啊
DNase还有RNase啊
结果破碎细胞以后离心得到的pellet,还有得到的复合物pellet都超级难resuspend的
一整块像Jelly的东西
能弄成大小碎块但是没有办法混匀
用针头反复吸吹也不行
加Uear到5M也不行
参考了一些提纯histone protein的protocol,
加HCl到0.25M也没有办法完全溶解
求建议! |
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z******i 发帖数: 120 | 45 仔细看了一下你的问题,你说的这个分别都挺好,混起来就沉淀,很常见。并非奇葩。
1.A B 是一样的BUFFER 吗? 不是的话,把A加到B的fuffer里有沉淀不?
2. 给A 和B 全加上SOLUBLE TAG, 比如 MBP 或者更强的 NUSA tag, 不相信你的蛋白
还会沉淀。结构是HELIX BUNDLE 吗?网上预测一下。
3. 蛋白结合DNA/RNA不? DNAse RNAse 不能除去很彻底。 要用PEI 和 硫酸铵。 另外
,高PI 很容易结合DNA/RNA, 并且在有GST存在的情况下有假结合。这个说来话长。 |
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l**********1 发帖数: 5204 | 47 Plant
There is reason to think that mRNA degradation sometimes starts with the
action of ribonuclease III, an enzyme specific for duplex RNA, which could
cleave in stem-loop structures and create sites for exonucleolytic attack.
RNase III is actually involved in the maturation of certain phage mRNAs as
they undergo posttranscriptional processing, but this involvement is not
known to occur with bacterial mRNAs.
PMID 17693527
Watkins KP et al.
A ribonuclease III domain protein functions in group I... 阅读全帖 |
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z*********8 发帖数: 1203 | 48 bme可以抑制RNase的activity,不过既然加了,我觉得是样品开始的时候就没有存储好
的感觉,你在liquid nitrogen里面snap freeze了么?或者你加RNAlater或者trizol了
么?其实,我个人感觉cell里面提RNA还算不是最难的,有些tissue是真心难! |
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l**********1 发帖数: 5204 | 49 For manipulating single strand RNA, LZ should use RNase free DEPC water for
the preparation of that 70% Ethanol. |
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T**********t 发帖数: 1604 | 50 DNase I不会degrade RNA。你有RNA degradation是因为你的sample有RNase污染。
DNase I处理之后跑胶纯化就不用管heat inactivation了,就是麻烦点。 |
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