f**u
发帖数: 346 | 1 我倒不这么看。
ChIP是故意把sonication的条件调到那样的，
binding site两边有那么一点点DNA可以确定在genome里的位置就可以了，
因为你希望最后结果是一个很尖的峰，如果DNA留得太长峰会变得不那么sharp。
这个并不是说sonication就一定会把DNA打得在binding site两边只剩那么短。
他们在做Pich的时候肯定把sonication的能量减小了，
打完的DNA平均长度肯定要大很多。 |
|
s******s
发帖数: 13035 | 2 //nod.
刚查了一下PiCH的protocol, ms确实不强，大概2-4kb,那么中间就应该有很多bind
ing site了 |
|
n********k
发帖数: 2818 | 3 you are referring to the traditional sequencing reaction...what about the
high throughput sequencing reaction(I am actually ignorant about here too:))
)...Also, I thought the RNA-Seq(the real one:))) doesn't need any
amplification of RNAs...so that would be a lot less than 0.1pM... |
|
s******s
发帖数: 13035 | 4 you are wrong.
RNA seq need amplification after addition of adaptors
)) |
|
n********k
发帖数: 2818 | 5 In a sense, you don't want the power too strong and the fragment too short
here...otherwise kind of lose the goal of developing the protocol... After
all I don't we aim to use this method to determine exact location of the
binding sites... |
|
n********k
发帖数: 2818 | 6 rather different...if you have an idea of what are possible candidates,
there are easier ways to sort it out...
size |
|
|
s******s
发帖数: 13035 | 8 恩。知道binding protein再回头找binding site就容易多了 |
|
n********k
发帖数: 2818 | 9 I am talking about the Single-Molecule RNA seq, directly use the RNA rather
than the RNA-seq(actually should be called cDNA-Seq) everyone is talking
about |
|
s******s
发帖数: 13035 | 10 don't Know that. do u mean pacbio? is their direct rna-seq commercialized?
rather |
|
n********k
发帖数: 2818 | 11 Then it came back to my initial question again, what's the real problem here
making it so hard...because we only have 2 copies of the binding even per
cells and that could be easily buried in background...or else??
If the sensitive is around 0.1pM, I wouldn't even think to try with any stem
cell work or mammalian cells....if it could go down by a factor of 50-100
or so, then it might be worth trying...even 10 is still not enough, still
take too many plates (600 plates of stem cells is like a jok... 阅读全帖 |
|
s******s
发帖数: 13035 | 12 看你有没有钱，你的project有没有意义了。
有高级的ms spec，可以sample量少100倍。不过你有劝那种实验室
帮你做ms spec的精力还不如养1000盘ESC
here
stem
least |
|
n********k
发帖数: 2818 | 13 I c...don't know, likely not yet...but I am talking about the potentials...
this is really an issue close to many hardcore TF/epigenetic people's heart.
.. not quite like the way many go like the Harvard lady you mentioned:)))...
.it is kind of like teaser--they just mess around it but never get to the
real sweet stuff... |
|
s******s
发帖数: 13035 | 14 最终理想，那个IBM还是GE的啥测序孔一类的，DNA拉一遍就知道序列了。
进化版本，chromosome拉一边，把所有epigenetic marker全读一遍。
heart.
.. |
|
n********k
发帖数: 2818 | 15 can u name some labs or type of MS which could do this? sounds very rare
ones?? |
|
n********k
发帖数: 2818 | 16 Sure..:) but I am not counting on them on the epigenetic marker stuff.... |
|
s******s
发帖数: 13035 | 17 没用心记。忘记是公司广告上的还是paper上的。很可能是paper上
看到的，哪个national lab专做ms的paper, 所以应该比较rare |
|
n********k
发帖数: 2818 | 18 it seems the tech is around the corner now(or maybe not:)), I just searched
online and it seems some are talking about single molecule MS now.... |
|
g****1
发帖数: 261 | 19
This is still a tough problem. I am aware that a few friends have been
trying the modified method reviewed here with modest success. You might get
some ideas there.
http://pubs.acs.org/doi/abs/10.1021/cb100294v |
|
g*********3
发帖数: 177 | 20 原来这么多人关心这个问题 呵呵。
其实某位说的ZNF的想法,我读了一篇paper,已经有类似的人做了,但是发的paper貌似很
一般.
至于MS的问题，昨天和老板讨论了下，fisher orbitrap 貌似能检测到<0.1pmol吧。
其实把多个copy转到chromosome里面去是个好方法，但是这个在动物身上就不好做了吧。
听完各位描述，顿时觉得phd得延期是必然吧。。。（或者毕业no publication） |
|
g*********3
发帖数: 177 | 21 这个有人做了。。。效果不是很好貌似。（转的质粒）
关键是你还得保证用于纯化目的的那段序列不会起到recruit TFs and GTFs...造成假
阳性 |
|
g*********3
发帖数: 177 | 22 谢谢。看到ACS比较激动，隐约感觉到这个问题得biophysics的东西帮助解决。
get |
|
g*********3
发帖数: 177 | 23 请问single molecule MS...能给个链接吗？
searched |
|
g*********5
发帖数: 2533 | 24 I am not professional... |
|
n********k
发帖数: 2818 | 25 Good one...Kind of what I have been going through although I am still not
giving up hoping something might happen with MS...anybody working with
microfluid stuff or if those thing could in any way help the sensitivity or
detection limit problem...any expert there? |
|
|
n********k
发帖数: 2818 | 27 BTW, the two PIs you worked are so weak...or they are not in the related
field?...to any reasonable one(even a good junior grad) in TF/epigenetics,
it is simply so obvious... |
|
f**u
发帖数: 346 | 28 谢谢信息。具体作过的和我们几个纸上谈兵的就是不一样。 |
|
i*****g
发帖数: 11893 | 29 我觉得，玩技术突破不是不可以做，
比如那个哈佛的yang shi，做histone demethylation的，就是靠TAP+ms，弄到一个新
蛋白
但是，我认为主流是玩story，making sexy story是主流,
functional study & disease
弄这些技术，突破，是次流，很容易辛苦很久，也没有什么特别的功能
这个没有办法，就类似：你认为美国股市应该崩溃，但大佬们不认为
人在江湖，生存第一 |
|
|
s******s
发帖数: 699 | 31 非常感谢，我又看了一下产品介绍，虽说是自带质粒，但只用加一种抗生素就可以了。
“The plasmid contains a functional partitioning locus that ensures
proper plasmid segregation, preventing plasmid loss even in the absence of a
selective agent.”
谢谢提示：） |
|
z*t
发帖数: 863 | 32 Beutler得奖，Medzhitov会不会有意见？
http://www.sciencedirect.com/science/article/pii/S1074761309002
Ruslan Medzhitov
This year marks the 20th anniversary of a publication (Janeway, 1989) that
in many ways revolutionized our understanding of the immune system. As part
of the proceedings of the Cold Spring Harbor Symposium on Immune Recognition
, the paper authored by the late Charles A. Janeway, Jr. was not a standard
peer-reviewed publication. In fact, Charlie used to refer to it as “the
best paper I've ... 阅读全帖 |
|
l**********1
发帖数: 5204 | 33 大鼠/欧洲野兔/水䶄/鸡 的MHC 是越外(性)交: 路边的野花 不采白不采) 好似后代越advantageous哦
欧洲野兔 MHC paper:
Homozygosity at a class II MHC locus depresses female
reproductive ability in European brown hares
PubMed link:
//www.ncbi.nlm.nih.gov/pubmed/20731776
Wiki:
//en.wikipedia.org/wiki/European_Hare
水䶄MHC paper:
Selection Maintains MHC Diversity through a Natural Population Bottleneck
//www.ncbi.nlm.nih.gov/pubmed/22323362
Wiki:
//en.wikipedia.org/wiki/European_Water_Vole
据说恐龙演化过来的鸟类中的鸡 MHC paper
PNAS (2011)
Th... 阅读全帖 |
|
m******5
发帖数: 1383 | 34 I have a question on this:
Thus said, the method proposed by LZ would be a very good strategy to
produce hypolymorphic allele? since most mRNA are subjected into non sense
mediated decay, so the protein level would be reasonably low.
And where to search for EGFP/LACZ trapped ES cell line? I searched around
but found only few available trapped locus, could you recommend some company
making good use of it?
level |
|
D*******8
发帖数: 4 | 35 Please don’t dig me out. I am writing this out of my conscience as a
scientist. We are using, largely, the tax payers’ money to explore
mechanisms underlying diseases that we don’t have a cure, yet. We are doing
this in the hope that little bit knowledge can accumulate till one day we
can find that magic bullet. However, there are people out there, their
purpose is to use these money to achieve their personal fames, sometimes
international ones, totally disregarding our original noble cause. I... 阅读全帖 |
|
p*****m
发帖数: 7030 | 36 不觉得他这个NGS的主意有什么太不对的 没有人能保证KO mice就真的只有一个gene
locus被改变了，free end DNA在细胞里什么事都能干。。rescue可以部分的说明
phenotype来自KO本身，但是不能完全说明，尤其当这个rescue其实本质上是over-
expression的时候
当然现实中基本上没有谁会去做个sequencing来说明自己的KO mice真的只有一个gene
被影响了 但是大家有意识不去做这个是一回事，这个验证本身有没有道理是另外一回事 |
|
y********a
发帖数: 138 | 37 you are way too literate about my statement.
I agree with you it is not WRONG to check target locus & neighbors and
even doing whole genome profiling to ensure no mis-interpretation of the
results.
I was laughing at the idea of thinking NGS is to the rescue of all biology
problems (although myself is intensively engaged in this area).
gene
回事 |
|
y********a
发帖数: 138 | 38 Fine, I already said I agree with that.
"I agree with you it is NOT WRONG to check target locus & neighbors and
I simply say it is funny to always think NGS first to solve a bio problem. |
|
n******7
发帖数: 12463 | 39 情况大概是这样：我们做了很多cDNA clone，测序之后选取了一些进行了下游的实验，
主要是in vitro 的protein实验。我们鉴定了很多新的splicing isoform，其中有不少
有premature stop codon(可以是splicing 造成的，也可以使indel造成的）
现在我们想做一些quality control，去掉一些不靠谱的transcripts，于是出现了分歧
组里的大姐想法是要尽量跟已知的protein一致。把每条isoform对应的protein align
到已有的protein sequence上。如果整条protein基本align上去，即便使truncated
protein, 不管什么原因，都可以作为partial protein 保留。但是如果shifted frame
的amino acid sequence长到一定程度，那就认为这些protein sequence跟已有的
sequence太不一样，要除去
我完全明白大姐为什么那么想，因为我们实际test的是一些protein sequences
但是就我这部分工作，我需要关... 阅读全帖 |
|
l**********1
发帖数: 5204 | 40 why only try BAC vector method only
alternatively you can try PB transposons vector
>Transfection of naked DNA has been used for large-cargo delivery.
Pronuclear injection of bacterial artificial chromosomes (BACs) has been
successful for transgenesis of up to 300 kb. However, the integrity,
integration site and copy number can not be controlled. BAC vectors have
also been used for targeting large cargos to defined genomic positions in ES
cells via homologous recombination (Valenzuela et al., 20... 阅读全帖 |
|
l**********1
发帖数: 5204 | 41 Exactly,
Please go to
//www.biotechniques.com/multimedia/archive/00010/01315rr05_10077a.pdf
it shown that complete genotyping of a floxed locus is possible with three appropriately placed primers
and that this triplex PCR can be performed simulta- neously with a universal PCR assay for the detection of
Cre transgenes.
[回复]
[ 8 ]
发信人: robin95 (robin95), 信区: Biology
标 题: Re: 请教用REAL－TIME PCR 测定基因KNOCKOUT 小鼠是－／－ 还
发信站: BBS 未名空间站 (Thu Feb 16 11:31:37 2012, 美东)
>非常感谢啊， 那就是说要３个PRIMER 一起加， 而不是像普通PCR 那... 阅读全帖 |
|
l**********1
发帖数: 5204 | 42 在说你Boss 吧?
Genetic screen 也有
笨的，比如直接在老鼠中筛表型。这类的，我不客气地说，你在得到mutation之前，你
的研究还没真正开始。你只是在做任何高中毕业生都可以做的。不信，你看你如果没有
mutation, 只有locus 或QTL，你能发在哪里？大概只能是Mammalian Genome之类的。
对我老人家来说，低于JBC 水平的，我都懒得写了。我的原则是宁吃仙桃一口，不啃烂
杏一筐。
http://www.mitbbs.com/article_t/Biology/31639187.html |
|
T****i
发帖数: 15191 | 43 前面有人竟然认为genetic screen是试错法。我老人家不得不本着为下一代负责的态度
，再聊一聊。
生物学研究，基本有hypothesis driven 和 fishing 两类。两者在过程中都不可避免
要试错。但试错不应该成为主流，否则就是我说的试错法。
Genetic screen，设计思路非常重要，对逻辑的严密和创造性都是考验。正式开始之前
，还要有proof of concept。顺便说一句，Peter Walter在yeast genetic screen 上
就非常牛。我佩服的五体投地。感兴趣的应该多看看他的工作。Genetic screen 也有
笨的，比如直接在老鼠中筛表型。这类的，我不客气地说，你在得到mutation之前，你
的研究还没真正开始。你只是在做任何高中毕业生都可以做的。不信，你看你如果没有
mutation, 只有locus 或QTL，你能发在哪里？大概只能是Mammalian Genome之类的。
对我老人家来说，低于JBC 水平的，我都懒得写了。我的原则是宁吃仙桃一口，不啃烂
杏一筐。
Biochemical fractionation 和 g... 阅读全帖 |
|
T****i
发帖数: 15191 | 44 我跟她工作了那么多年，我对她什么水平没有感觉？她肯定读文章，但没有系统读。多
数时候就读读abstract而已。PI到她那份上，更多的是networking 了。
我的例子里，她已经让那个听话的薄厚废掉了。8年只发一篇文章，还是二流的，就因
为她瞎指挥。试了一个又一个没有经过仔细推敲的想法。有的时候连hypothesis 都算
不上，因为没有足够的证据或逻辑来支持。举个例子，但我不能说的太具体，因为人文
章还没发呢。该薄厚的项目曾经有一个technician 帮忙，找到Gene A 一个genetic
modifier，mapped 到一个locus，有几个基因，恰好有一个gene B 跟oxidative
stress和DNA repair有关。该薄厚又发现在culture neuron里，该蛋白B有一小部分在
线粒体上。然后我前老板就非常兴奋，立刻认定是线粒体功能受影响。就订了一台测老
鼠呼吸的仪器，做了一番发现呼吸耗氧量没变化。然后就认为可能只局限于神经系统，
于是订了很多抗体，kit，都是关于线粒体的。结果什么变化都没有。我没时间讲太多
细节。此处略过两年。。。后来他们意识到也... 阅读全帖 |
|
a*****x
发帖数: 901 | 45 更快的方法是用Talen或者ZFN knock-in GFP，我们做过不会影响ES分化的。还可以
knock-in在gene A的locus同是做knockout,这样连knockout老鼠都不用了。
GFP
gene |
|
s******a
发帖数: 472 | 46 这个triplex PCR确实设计很巧妙啊。。。
不过不适合我的问题，如果要证明我的transgene（insert）整合到特定位点，应该把
引物一个设计在transgene上一个在homologous arm外面做muhe提到的long range PCR。
这样至少能保证transgene在targeted locus整合。 |
|
l**********1
发帖数: 5204 | 47 please go to
//evol.mcmaster.ca/cgi-bin/my_wrap/brian/evoldir/PostDocs/
then search web with key word:
Droso
match three
then click just one:
Postdoctoral Researcher in Drosophila Evolutionary Genomics
Potential start dates are between April 1 and July 1, 2012. Please
indicate the earliest start date you would consider.
Feel free to contact me with any questions (jpool (at) wisc.edu).
John Pool
Assistant Professor
Laboratory of Genetics
University of Wisconsin-Madison
jpool (at )wisc.edu
----
A... 阅读全帖 |
|
b******s
发帖数: 1089 | 48 既然这几天谈到了Zhu Jian-kang，Deng XW和Dong XN。我们可以从他们开始谈起。
如前所述，我没有大家喜闻乐见的8卦素材，所以基本上我这里只是列出他们从学生
时代起的publication等，大家可以看到他们如何在career上有个大的跳跃。
Zhu Jian-Kang
教育背景
Beijing Agricultural University B.S. 1987 Soils & Ag. Chem
University of California, Riverside M.S. 1990 Botany
Purdue University PhD. 1993 Plant Physiology
Rockefeller University Post-Doc 1994 Molecular Biology
博士老板属于中牛，但是看Zhu的访谈，应该对他很欣赏，对他的事业应该很有帮助。
Zhu薄厚只做了一年，而且没有文章。应该是在开山老怪Nam-Hai Chua那里做的。
职位：
2010-Present Professor of Horticulture and Bioche... 阅读全帖 |
|
b******s
发帖数: 1089 | 49 (1)
既然这几天谈到了Zhu Jian-kang，Deng XW和Dong XN。我们可以从他们开始谈起。
如前所述，我没有大家喜闻乐见的8卦素材，所以基本上我这里只是列出他们从学生
时代起的publication等，大家可以看到他们如何在career上有个大的跳跃。
Zhu Jian-Kang
教育背景
Beijing Agricultural University B.S. 1987 Soils & Ag. Chem
University of California, Riverside M.S. 1990 Botany
Purdue University PhD. 1993 Plant Physiology
Rockefeller University Post-Doc 1994 Molecular Biology
博士老板属于中牛，但是看Zhu的访谈，应该对他很欣赏，对他的事业应该很有帮助。
Zhu薄厚只做了一年，而且没有文章。应该是在开山老怪Nam-Hai Chua那里做的。
职位：
2010-Present Professor of Horticulture and Bi... 阅读全帖 |
|
l**********1
发帖数: 5204 | 50 Off-target 中奖拉 搂住的那单locus siRNA
还是考古下上个月的 旧贴 如何
同主题阅读：现在发文章还需要2套siRNA吗？
[版面:生物学][首篇作者：CAMS] , 2012年06月19日
answer:
一般至少要3 different oligos才能自己放心
siRNA的off target是非常严重的
最严谨的是shRNA KD,然后rescue with ectopic expression
shRNA也需要2-3个independent clone to exclude clonal effect
http://www.mitbbs.com/article_t/Biology/31683985.html
knockdown |
|
