n*******e
发帖数: 62 | 1 Abstract
1 – “Rose is a rose is a rose is a rose”, C# is case sensitive, that
sucks.
2 – The Switch clause
3 – Event-Handling code
4 –Stupid symbols
5 – Autocorrection in Visual Basic actually works
6 – Lack of supported functions, such as:IsNumeric,PMT, etc. but they don’
t exist in C#.
7 – That wretched semi-colon.Why do I have to end every line in C# with a
semi-colon?
8 – Arguments and variables.The order of words in a C# variable declaration
is wrong.the C# method of having to prefix argume... 阅读全帖 |
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R*s
发帖数: 2041 | 3 example:
--------------------- ----------------------------
| bait | activator| + |fragment A | reporter gene |
--------------------- ----------------------------
Bait ait is the gene u r intestered in, and is attached to a
transcription activator. fragment A is one of those from a DNA library
which is attached to a reporter gene.
Basically you transfect both the bait construct and library construct
into yeast (a and alpha respectively) and let the mate (each yes... 阅读全帖 |
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n***h
发帖数: 11 | 4 照我的记忆说来：
yeast 2 hybrid system是两个蛋白之间的binding
yeast 1 hybrid是蛋白质和DNA之间的binding ，就象ras说的，测出的是bait和
fragment之间的结合，可以象ras说的那样fragment是library, 来找和已知蛋白
结合的特定element，也可以特定的fragment，转cDNA library进去，找和这个
fragment 结合的蛋白。 （infact that's what I did)
回来说2hybrid。transcriptional factor一般至少有两个domain: binding domain
和activition domain. binding domain binds to DNA elements, while activation
domain activates the transcription of downstream genes. 我们要测试蛋白A
和蛋白B是否bind, 就做以下两个construct：
—————————— —————————... 阅读全帖 |
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b*z
发帖数: 48 | 5
yes/no.
a gene directs the synthesis of one polypeptide. a protein may
contain more than two different polypeptides and require more
than two genes.
in some cases a gene may contain more than two introns (inter-
rupted sequences) and exon (expressed sequences). an RNA
transcribed from such a gene may undergo splicing, that is, the
re-combination of introns and exons, resulting several mRNAs from
one single gene. these mRNAs may then be translated into a group
of proteins. usually such proteins |
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n*******e
发帖数: 27 | 6 they are basically the same. the principle of run-on is that you first
isolate the nuclei and remove most of the proteins, but still keep the
RNA polymerase associated with nascent transcribed target gene. then
when you add NTP and p32 labeled UTP, the TXN will run on and then all
the product will be labeled. it allows mapping of the start point from
the size fractionated products.
run-off means that TXN occurs to in vitro DNA fragments, which are
typically restricted fragments with the start si |
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b***g
发帖数: 110 | 7 做植物的最早发现了一种叫做corepression的现象，这种现象按我的理解是，就是用转
基因的方法去rescue一个phenotype,结果却产生了一种loss of function的phenotype.
当时植物学家无法解释这个现象，而且做corepression还要generate transgenic
lines,其实挺不方便的。后来做其他organisms也发现了这个现象，但都不知道机理。
好，现在让我们跳到1995年.康乃尔大学ken kemphues实验室研究的一个非常聪明的中
国学生，Guo Su（现在UCSF), 发表了一片cell的文章。在这篇文章中，她做了一个
antisense phenotype copy analysis,就是in vitro transcribed antisense RNA of
par-1,然后直接注射到c.elegans gonad,希望能够knockdown par-1 activity.事实上
，她成功了。但是更有趣的是，当时她的control是sense strand of par-1 RNA,这个
control却产生了同样 |
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s******e
发帖数: 370 | 8 就是说cre由卵子提供要保险一点，因为受精的时候卵子里已经有active cre了，这样
进去的精子的chromosomes立刻可以被process。如果是在精子里的话，它还需要时间
transcribe
不过不管怎样F1出来以后还都得再筛一次，选完全的KO去establish line |
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s******e
发帖数: 370 | 9 我们做的是complete KO， 不是tissue specific，所以要从卵子开始就得有active的
cre——跟上面各位不同哈
F1出来，PCR，碰到过既有被cre切过以后产生的小带，又有未切的cKO带，就把这个叫
partial。究其原因，猜想可能是cre被transcribe出来的时间太晚又太短，受精卵分裂
之后有些细胞带了cre有些没有。
但是即使是partial，如果它的生殖细胞是KO，我们还是可以用它来establish line的。
tissue specific暂时还没有做到⋯⋯ |
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H****N
发帖数: 997 | 10 It seems to me that your western is not reliable. As to the RT-PCR, you
should use primers spaning the dedleted exon to check that exon is actually
gone. Remember, after deleting one exon, the rest of the gene can still be
transcribed and make a deleted mRNA.
member |
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a****o
发帖数: 1786 | 11 Is it possible to synthesize a short RNA oligo with NH2 at 5', anneal this
oligo to your single strand DNA template, add T7 RNA polymerase to
transcribe. The yield may be low, but the incorporation efficiency should be
really high. :))) |
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a******g
发帖数: 129 | 12 RT-PCR有时候是看不出这个gene是不是表达的，因为你的primer只探测一部分mRNA的表
达，如果你用RNA-seq去看的话，你会发现这个gene的某些exon highly transcribed,
但有的几乎没表达，结果就是这个gene的mRNA不完整无法被translate,你自然就看不见
蛋白了 |
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M*****n
发帖数: 16729 | 13 the central dogma of developmental biology is that genes (DNA)are
differentially transcribed and expressed in different types of cells and
hence confer these cells different fates and functionalities. this is under
the assumption that the genome (genes on DNA level) remains the same in all
the cells in that oragnism.
but this is not true in reality. the genome in every cell is not all identical.
there will be rearrangement such as in B cells. there will be copy number
variations such as in the p... 阅读全帖 |
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T**********t
发帖数: 1604 | 14 不同表达（一个表达，另一个不表达）
意思应该是一个transcribe成了mRNA，一个没有。这个应该是用的RNAseq技术，测的是
不同organ里的mRNA吧。 |
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z***q
发帖数: 907 | 15 就是说结构不稳定主要是端基没有GC的原因了？
我曾经做过一个mutation,就是把端基的2个UU都去掉，再把邻近UU的AU变成GC，这样就
有2个GC连在一起，可以transcribe了。然后发现得到的样品dimer有大概30%-40%，这
是不是说明不仅stem,其实loop也不稳定？
非常感谢！
~~~~~~~~~~~~~~~~~~~~~
这个我感觉也没什么用 |
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M*******s
发帖数: 108 | 16 there are transcriptome data
which TF will transcribe which gene, and also combinatorial effects of TF.
you can just use that kind of data to pull out your genes then you may get
TFs
but I doubt it is convincing enough for publish if without ChIP data
you may carry some further study. For instance, build up the regulatory
network first, then find the dysregulated genes from upstream to downstream
in the network, then you may find some trace of the dysregulation. Generally
, upper-stream usually ... 阅读全帖 |
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m***o
发帖数: 272 | 18 Thanks, whitesand.
1. We are thinking of doing that way. But most plasmid has a strong promoter
, I am guessing whenever they meet ATG they all will transcribe. The other
possible is we clone this gene's own promoter and mutate each of the 3 ATG
to see. There will be a lot of work and now I do not have any idea how long
the promoter will be.
2. If my GSP recoginze a sequence in the reverse orientation, why it
transcript the complete exon not part of it?
I am thinking of what wenqian said because... 阅读全帖 |
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x********u
发帖数: 430 | 19 The procedure I found in the literature;
1) grow E. coli to late-exponential phase
2) extract total RNA with RNeasy Protect Bacteria Mini Kit(Qiagen)
3) purify RNA by RNeasy Mini Kit(Qigen) spin column
4) send samples to xxxxx University Functional Genomics Core Facility for
amplication, labeling and hybridization to the Affymetrix GeneChip E. coli
genome 2.0 Array.
5) Data analysis by MatLab bioinformatics toolbox
Question: For amplication of total mRNA and labeling of the resulting cDNA
in ste... 阅读全帖 |
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a********k
发帖数: 2273 | 20 花了一个小时，深深的鄙视一下自己的无聊行径！！
125 蔡亮 男 1980年11月 复旦大学 生命
科学 2007年12月毕业于[美国]北卡大学 [美国]加州大学旧金山分校 博士后
Cai L, Mostov K. Polarity is destiny. Cell. 2009 Nov 13;139(4):660-2. PubMed
PMID: 19914162; PubMed Central PMCID: PMC2900917.
Cai L, Makhov AM, Schafer DA, Bear JE. Coronin 1B antagonizes cortactin and
remodels Arp2/3-containing actin branches in lamellipodia. Cell. 2008 Sep
5;134(5):828-42. PubMed PMID: 18775315; PubMed Central PMCID: PMC2570342.
Cai L, Makhov AM, Bear JE. F-actin... 阅读全帖 |
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n********k
发帖数: 2818 | 21 I'll answer as I know:
5p/3p: miRNA transcribed from 5 prime/3 prime(essentially the 5p and 3p are
antisense to each other).
The family member or clusters are case by case I believe(not sure though).
Some are very similar, sharing the seeds sequences while the other are not
but within the same genome regions.
siRNA are completely matched/complementary to their targeting sequences---
used to be only for exogenous but now we know there are endogenous ones as
well.
small RNA means small RNAs--sma... 阅读全帖 |
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l**********1
发帖数: 5204 | 22 ZZ
老外的forum 有关的讨论帖 的最精彩的一句推论:
>So if I understood well, you suggest to lengthening the 5' end of the
construct including part of the promoter in a promoterless vector and then
transfect different cell lines and see in which one the entire transcript
will be expressed.
details pls go to
htp://molecularbiology.forums.biotechniques.com/viewtopic.php?f=2&t=31288
看看能否有助LZ的这问题的解决?
有一点务必牢记: without in vivo experimental data support for a new species 5'UTR information Blast
information is just a block ... 阅读全帖 |
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l**********1
发帖数: 5204 | 23 RE LZ
just go to
htp://www.mitbbs.com/article_t/Biology/31627067.html
11f
一句话： 要确定某个表达蛋白(A物种) 的真正的5' UTR 范围碱基序列
最好将其包括启动子部分的碱基序列转接到一个非启动子功能的载体
然后转染到异种(B物种: 可以是A物种的近缘) 的Cell line 表达那个目标蛋白
再提取那个表达后的目标蛋白
如果后者同In vivo (A物种) 的生化物理性质完全一致,
那么其包括启动子部分的碱基序列 就是真正的5' UTR 范围碱基序列
[回复]
发信人: lotkaeuler11 (Fibonacci), 信区: Biology
标 题: Re: 急求：怎样从基因组序列中找出基因的5'UTR确切序列？
发信站: BBS 未名空间站 (Thu Jan 19 05:09:42 2012, 美东)
ZZ
老外的forum 有关的讨论帖 的最精彩的一句推论:
>So if I understood well, you suggest to lengthening the 5' end of th... 阅读全帖 |
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A********2
发帖数: 107 | 24 Should be transcribed in the same direction as replication since they are
highly expressed genes |
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B****n
发帖数: 22 | 25 the cell survived your selection. so your lentivirus works fine.
you may extract the DNA from the infected cell to see if your cDNA is still
there or not.
If i remember correctly, when you transfected 293T cells, the region between
the 2 LTR region will be transcribed and spliced. when the lentivirus
infects cells, the virus genome(RNA) will be retrotranscribed into DNA and
integrated into genome. So if there is any intron or intron like structure
in your contruct, it maybe processed as an intro... 阅读全帖 |
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o********r
发帖数: 775 | 26 今天的nature news:
http://www.nature.com/news/rna-studies-under-fire-1.10502?gobac
RNA studies under fire
High-profile results challenged over statistical analysis of sequence data.
Erika Check Hayden
25 April 2012
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High-throughput RNA sequencing has yielded some unexpected results in the
past few years — including some that seem to rewrite conventional wisdom in
genetics. But a few of those findings... 阅读全帖 |
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c********e
发帖数: 598 | 27
However,
no
now
in
1) how many clones did you test from one sample?
2) Are these 400 mutated genes transcribed? |
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c*****g
发帖数: 66 | 28 像你这种情况，
1. 找个commercial的软件做分析，最后给你output一个list of differentially
expressed/spliced/transcribed genes。很多大学的core facility可能有这样的
license，你们可以免费的用。这个方法是最好的，你甚至可以利用他们的技术支持给
你解决很多问题。
2. 你的facility既然都给你做mapping了，顺便让他们给你们call一下differential
expression，应该就是一两百块钱的事。
3. 用galaxy （galaxy.psu.edu），这个需要花的功夫比1.多，不过肯定比学什么编程
之类的少多了。galaxy的主页上有很多tutorial。基本上主流的软件都可以在galaxy上
用，等你熟悉了流程，是相当快的。只要你不是很复杂的实验设计，大部分的软件只要
accept default就行了。
很多给你的帖子回复的都是bioinformatician的mind set，不理解biologist的需要。
其实你需要的就是一个list，最多加上什么pathway a... 阅读全帖 |
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j****x
发帖数: 1704 | 29 相对于HCV研究，即便是美国，HBV的经费也并不少。只不过相当大一部分经费是划分在
了liver cancer，liver tranplantation等等临床相关领域中，而真正HBV的所谓基础
研究，因为你所说得包括研究模型的各种限制，长期致力于这个领域的课题组并不多，
还有一部分是从HIV那边过来打酱油的，因为都属于Retro-transcribing viruses。感
染率低是实事，但要说因此而甚请不到经费，就有点本末倒置了。看看ASSLD每年的年
会，就很清楚的。此外，在美国感染率更低的病毒甚至早已绝迹了天花病毒，研究经费
也同样不少。
德法在肝炎病毒领域的研究确实有传统，不仅仅是HBV。海德堡的Ralf Bartenshlager
在HCV领域的地位，绝不输于Rice，对于目前多种antiHCV新药研发成功的贡献，无出其
右者，也是因为解决了HCV体外复制模型这一基本难题。
李博士的成果对于国内的课题组而言意义更大。原因很简单，之前唯一的感染细胞模型
即HepaRG细胞系，对中国地区是禁售的，换言之，就算你出1万欧元你也买不到（国内
能出这个钱的组不在少数，但人家就是不卖给... 阅读全帖 |
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z*******6
发帖数: 679 | 30 为啥要不通过rRNA呢？...
如果是in vitro transcribed的，那也能看到一条清晰的带... |
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l******a
发帖数: 3339 | 31 我真服了，都是高人啊，我想知道promoter 会transcribe吗？ |
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l******a
发帖数: 3339 | 32 我真服了，都是高人啊，我想知道promoter 会transcribe吗？ |
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B******o
发帖数: 496 | 33 The vector does look weird. First, what is the promoter for the viral
expression. Usually, if it is MMLV, then the promoter is contained in 5'LTR,
so the viral genome is transcribed from there. Otherwise, people will put a
CMV in front of 5'LTR to express it. Without a promoter, the viral genome
will not be expressed and packaged into virus particles.
Second, I never saw anybody inserted Ori within a viral genome. It is very
weired. Not sure how it will affect your gene expression.
Third, poly A... 阅读全帖 |
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L*******a
发帖数: 293 | 34 我觉得这个会是一个热点。一方面是因为technologically practical，技术进步让大
家能够从仅研究一维/线性的调控扩展到研究三维/长程的调控；另一方面三维调控本身
是个biologically interesting的问题。cohensin，CTCF这些调控因子影响到细胞核内
subdomain的形成。从线性角度的chromatin regulator binding在更高的维度上有long
range interaction。Actively transcribed基因在三维空间上互相靠近，形成co-
transcription中心。类似的，facultative heterochromatin里的repress基因也会相
互靠近，叫polycomb body。是higher-order chromatin structure水平的调控。
seq |
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m******g
发帖数: 467 | 35 PNAS 14.03.11
推荐：
1. Human coding RNA editing is generally nonadaptive
稍微了解一下：
1. High-throughput sequencing reveals inbreeding depression in a natural
population
2. Evolutionary history and metabolic insights of ancient mammalian uricases
allowed ancient frugivorous apes to rapidly convert fructose into fat
小知识：
1. Homology-directed repair of DNA nicks via pathways distinct from
canonical double-strand break repair
HDR at nicks, but not DSBs, is associated with transcription
8-fold more effici... 阅读全帖 |
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g********6
发帖数: 86 | 36 in vitro: 细菌表达GST融合蛋白，做GST pull down，可以用你提到的in vitro
transcribed rRNA，33P标记，完了之后做液闪。
in cell lines：转染HA/FLAG标记的蛋白表达质粒，做RNA immunoprecipitation，然
后做qPCR或RNA-seq。前面有人提到用CRISPR敲进标记，我觉得可能比较麻烦，不如用
质粒来的简单。当然最可信的方法还是用目的蛋白的抗体。 |
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n********e
发帖数: 72 | 37 UTR只是不translate，但是是被transcribe的，当然是EXON |
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g*****n
发帖数: 250 | 38 There is nothing unusual if both anti-sense and sense probes light up. Some
genes do get transcribed from both strands. The sense and anti-sense
transcripts can be in the same tissue, but may or may be in the same cell. |
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