o********r
发帖数: 775 | 1 以前有那种定点mutagenesis的protocol，用含mutation的primers和high fidality的
polymerase做linear amplification，然后用只切methylated DNA的RE处理，再
transformation。这样以后想怎么用都行。
现在不用这个protocol了？ |
|
w***e
发帖数: 269 | 2 Here is the problem. We recently developed a mouse knockout line with a
company. The entire gene is knocked out with all the exons and introns
being replaced with a reporter gene (lacZ) cassette. We got the
heterozygous (+/-) mice and I did the breeding. But now I have trouble
genotyping to find out the knockout mice (-/-). I tried two pairs of
primers in that gene to PCR out ~400 bp fragments in the last exon (the
biggest exon which codes the functional domain of the protein). PCR with
one pai... 阅读全帖 |
|
Z******5
发帖数: 435 | 3 前几天刚用invitrogen的5´ RACE System for Rapid Amplification of cDNA
Ends, Version 2.0搞定一个13kb的RACE出来。
RNA质量很重要；要是只做反转录的话，invitrogen的SuperScriptTM Ⅲ First-Strand
Synthesis System可能更好；可以考虑在3'UTR位置多设计几条Gene Specific
Primers，效果比Oligo dT应该要好很多；多设计几条nested PCR引物，进行多轮扩增。 |
|
n********k
发帖数: 2818 | 4 Hey, folks:
We are planning to perform some ChIP/RNA-Seq as well as miRNA array
analysis some time soon. I was wondering any of you could kind of share
some of your experience in general.
Thanks a lot.
Something like: which services do you use(in house, or company), software,
etc etc. Which protocols, Kits? Pros and Cons, critical steps and tricks...
how many reads do you have, is it enough to cover the genome or enough for
the purpose? replicates? what about mRNA v lncRNA v miRNA, do y... 阅读全帖 |
|
m***T
发帖数: 11058 | 5 Array原来的优势主要体现在cost上，但现在这个优势越来越小。NGS比起array有不少
优势，如throughput，coverage等，但也还是biased（虽然比array好），主要还
是因为需要in vitro amplification的原因。从理论上讲，3rd gen应该是最好的，但
技术上还不太成熟，error rate高，throughput低等等。
NGS的一个主要的hurdle是这个贴子的主题--数据分析方面，所以从这个角度上来看，
接下去的几年内对这方面的人才要求应该是比较高的，不管是academia还是industry。 |
|
o********r
发帖数: 775 | 6 NGS在library construction阶段有amplification，不过也因此引入了GC bias之类的
问题 |
|
m***T
发帖数: 11058 | 7 我上面的贴子已经提到了，二代技术基本上都要in vitro amplification
，这是二代技术导致偏差的主要问题。三代从理论上讲不用扩增从而避免了这项，但目
前技术上还不够成熟，错误率太高而且throughput也低。 |
|
e*r
发帖数: 103 | 8 请问有人用过1.5 round amplification kit吗？效果如何？ |
|
g*********d
发帖数: 233 | 9 科学家呼吁关注全球基因组数据库污染
样品处理有可能是导致DNA数据库广泛污染的最主要原因
2月16日发表在《公共科学图书馆·综合》（PLoS ONE）期刊上的一份研究报告称康涅
狄格大学的遗
传学家Mark Longo及同事发现由顶级公共测序机构提供的测序结果构建的基因组数据库
中的大约
1/5的细菌、植物和非灵长类动物基因组数据受到了人类DNA的污染，样品处理有可能是
导致DNA数据
库广泛污染的最主要原因。这一研究报告引起了生物研究人员及各大权威媒体的高度关
注，《科学
家》（The Scientist）杂志以及《自然》（Nature）杂志均在其官方网络上第一时间
对这一事件
进行了报道。
Mark Longo等在报告中呼吁科学家们需更加努力以确保测序获得的基因组不受到污染，
并应对来自
公共基因组数据库的基因组进行潜在污染检测。
“基因组污染是一个大问题，但却不是一个新问题，”加州大学进化生物学家、美国能
源部联合基因组
研究所系统发育基因组学计划负责人Jonathan Eisen说：“这篇论文或可帮助提醒人们
注意这一问
题。”
污染有可能在测序的任何一个阶段导入到基因组序列中... 阅读全帖 |
|
w******e
发帖数: 1187 | 10 Expert Opinion on Medical Diagnostics
Aptamer amplification: divide and signal
December 2008, Vol. 2, No. 12 , Pages 1333-1346 (doi:10.1517/
17530050802562016
)
Supriya Pai1 BS, Ana Roberts1 BS & Andrew D Ellington
2.Expert Opin Drug Discov. 2011 Jan 1;6(1):75-87.
Strategies for the discovery of therapeutic Aptamers.
Yang X, Li N, Gorenstein DG.
email: q************[email protected]
Thank you! |
|
c****l
发帖数: 1086 | 11 不好意思，没有暴露你的意思，也没有人肉的兴趣，
我只是本身对PDGFRA的功能，以及MSC 二者都感兴趣，这个paper刚刚出来的时候很是
兴奋了一阵，我们主要是对其在脑子里的功能感兴趣，PDGFRA在脑子里比较特异的在
oligodendrocytes progenitor cells 里表达， 在glioblastoma里有amplification，
所以我比较关注。
Philippe Soriano很牛的。有前途！ |
|
c****l
发帖数: 1086 | 12 不好意思，没有暴露你的意思，也没有人肉的兴趣，
我只是本身对PDGFRA的功能，以及MSC 二者都感兴趣，这个paper刚刚出来的时候很是
兴奋了一阵，我们主要是对其在脑子里的功能感兴趣，PDGFRA在脑子里比较特异的在
oligodendrocytes progenitor cells 里表达， 在glioblastoma里有amplification，
所以我比较关注。
Philippe Soriano很牛的。有前途！ |
|
h**********r
发帖数: 671 | 13 我在实验室用的Thermo，貌似work的不是很好。
Yeast DNA Extraction
(The protocol was modified by housepainter on December 28, 2009 according to
manufacture’s instructions)
Yeast DNA Extraction Kit, Thermo Number Description: 78870
Storage: Store at 4°C; if precipitant has formed, gently warm DNA Releasing
Reagents A and B at 37°C for 1-5 minutes.
Kit Contents:
Y-PER Reagent, 25 ml
DNA Releasing Reagent A, 20 ml
DNA Releasing Reagent B, 20 ml
Protein Removal Reagent, 10 ml
1. Pellet a 10 ml S. cerevisiae culture gr... 阅读全帖 |
|
c****l
发帖数: 1086 | 14 Lots of examples in the situation of amplification, for instance EGFR etc
RTKs |
|
c****l
发帖数: 1086 | 15 你如果实在讨论在体外的transformation的话，那就是和体内的概念完全不同了。
只有一个oncogene而且还是wt，（也就是还不能称oncogene只是proto-oncogene，），
即使是真正的所谓oncogene（mutated one）经典的象myc,ras，E1A on its own也无法
transform cellline，原因很明显，scott lowe，John Cleveland十多年前就发现了，
单独的oncogene acitvation会induce cell death。 必须要结合inactivation of P53
or Arf ...才可能transform cell。你所指的cellline往往都是immortalized，而
immortalized celllines 30% has p53 mutation, 30% has Arf mutation, another
30% has mdm2 amplification。
你要是真要找一符合你的标准的wt的“oncogene”，那我就告诉你一个吧。
SV40， 哈哈哈哈哈哈哈... 阅读全帖 |
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|
|
e**s
发帖数: 513 | 18 目前的理论好像是说，duplication的动力之一就是防备拷贝的时候出错的。至于多拷
贝的，最有名的就是ribosomal RNA gene了，官方的解释是说，没有translation的第
二轮的amplification，所以学要很多拷贝。
（病毒之类），而重要的东西很少能看到拷贝？ |
|
b*****u
发帖数: 75 | 19 (3p is the guy who has worked a lot on PTEN in cancer)
The Shang Nature paper kind of propose 1, PAX2 is a transcription target of
ER, 2. PAX2 is positively correlated with tumor growth.
However, the 2008 Nature paper says that PAX2 is a co-repressor of HER2 gene
transcription, which is tyrosine receptor kinase implicated in malignancy
transformation. I am not sure about HER2 amplification or any functional
roles in endometrical cancer. But still the two papers' conclusions seems
very different. |
|
w******e
发帖数: 1187 | 20 是有很多工作可以做，但如何入手就是另一回事了呵呵。
比如说，我觉得ultimate goal for affinity reagent development是thermostable,
self-integrated with detection & amplification mechanism, adaptable into
multi-target platform, tunable affinity under enviromental cue，tunable
density to achieve multivalent interaction.
那么怎么去实现这个目标？我脚的on paper aptamer其实是最好的candidate，但
过去二十年都被技术瓶颈限制着呐。最近的breakthrough能把boundary push到
多远？我没本事判断。类似的，protein engineering在thermostability上，
在integrating detection module/scaffolding module上，在molecular
switchin... 阅读全帖 |
|
n********k
发帖数: 2818 | 21 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 | 22 you are wrong.
RNA seq need amplification after addition of adaptors
)) |
|
a********k
发帖数: 2273 | 23 搜了一下，觉得院士还行嘛，很多人引用都还不错。
科学院院士饶子和，工程院院士曹雪涛，科学院院士常文瑞，科学院院士杨焕明，科学
院院士张启发
饶同学：The crystal structures of severe acute respiratory syndrome virus
main protease and its complex with an inhibitor,引用297
曹同学：Splenic stroma drives mature dendritic cells to differentiate into
regulatory dendritic cells，引用222
常同学：Crystal structure of spinach major light-harvesting complex at 2. 72
resolution，引用617
杨同学有很多，不过估计有人不齿他：A draft sequence of the rice genome (Oryza
sativa L. ssp. indica)引
用1953
张同学也有很多，挑一个小paper吧：Pat... 阅读全帖 |
|
s******r
发帖数: 2876 | 24 那就是说饶毅还没有到一骑绝尘的地步，
搜了一下，觉得院士还行嘛，很多人引用都还不错。
科学院院士饶子和，工程院院士曹雪涛，科学院院士常文瑞，科学院院士杨焕明，科学
院院士张启发
饶同学：The crystal structures of severe acute respiratory syndrome virus
main protease and its complex with an inhibitor,引用297
曹同学：Splenic stroma drives mature dendritic cells to differentiate into
regulatory dendritic cells，引用222
常同学：Crystal structure of spinach major light-harvesting complex at 2. 72
resolution，引用617
杨同学有很多，不过估计有人不齿他：A draft sequence of the rice genome (Oryza
sativa L. ssp. indica)引
用1953
张同... 阅读全帖 |
|
n******3
发帖数: 14 | 25 The Stem Cell Research Center at Roger Williams Medical Center in Providence
, RI is currently
seeking to invite a new research associate (or post doctor) to investigate
the usage of stem cells
in diabetes treatment both in vivo and in vitro.
Qualifications: Applicant should have either a master's with at least 2
years of laboratory
research experience or Ph.D’s degree. A broad knowledge in molecular and
cellular biology
is required as well as experience with virus replication for DNA
amplificat... 阅读全帖 |
|
L*****o
发帖数: 48 | 26 I myself never tried COUP-TFII antibody, just read about it from papers.
However, about EphrinB2/4 staining, you can try tyramide signaling amplification followed by antigen retrieval.
|
|
n********k
发帖数: 2818 | 27 I did the ChIP part and the core prepared the libraries and I believe it was
the same protocol for all samples. Apparently TF groups have much lower
amount of DNA to start with and I was wondering whether that would make the
background noise much big an issue and thus the percentage of the mappable
reads is very low. I have three conditions: the percentage of the mappable
reads is decreasing as the same way the starting amount of ChIP-DNA does. I
was wondering whether over-amplification could ... 阅读全帖 |
|
j*p
发帖数: 411 | 28 people use cell numbers from 10-100M to do normal TF chipseq. New
techniques are developing to chip in small amount of cells, as few as ~ten
thousands, as someone claimed. (for example: Single-tube linear DNADNADNA
amplification (LinDADA) for robust ChIP-seq) |
|
n********k
发帖数: 2818 | 29 This is so great and thank you very much.
1. the Antibody is at least decent---this gene has been chipped many many
times by many labs; and I confirmed the ChIP using QPCR...
2. I have been suspecting we may have the library overload problem or over-
amplification issue(if that makes sense). The core really followed the
histone protocol and was meant to get 20-30M reads, and it did for Histone
markers and Input DNA. However, for my TF, the 1st is 9M with 9% mappable
reads (I expect less bind ev... 阅读全帖 |
|
A********2
发帖数: 107 | 30 大猩猩跟人离得还是比较远吧，黑猩猩跟人最近，whole chromosome amplification？ |
|
y****6
发帖数: 196 | 31 try Tyramide amplification (Perkin Elmer) |
|
w******e
发帖数: 1187 | 32 因为sensitivity跟signal amplification有关。
btw WB的LOD通常是多少？pM还是nM？ |
|
w******n
发帖数: 371 | 33 标 题: 第一批CLS博士后基金入选者名单公示（清华博士后水准高）
发信站: 水木社区 (Sun Nov 27 11:44:48 2011), 站内
八位申请人拟入选CLS清华博士后基金，现将名单公示如下：
特等博士后基金：
1.龚蓉：2011年7月，在中国医学科学院北京协和医学院获得理学博士学位
2005年7月， 在武汉大学获得理学学士学位
代表性一作论文：
1.Rong Gong*, Ji Hu* Cheng Ding* et al. (2011). Role for the membrane
receptor guanylyl cyclase-C in attention deficiency and hyperactive behavior
. Science 333:1642-1646. (*equal authorship.)
2、梅子青：2009年12月，在清华大学获得理学博士学位
2002年7月， 在吉林大学获得理学硕士学位
1999年7月， 在吉林大学获得理学学士学位
... 阅读全帖 |
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k*****d
发帖数: 12 | 34 用SNP array找copy number variation算不算genomic cancer study? 在这一期的
Nature上（Nature. 2008 Oct 16;455(7215)）有四篇文章讲到发现neuroblastoma 有
ALK mutation，其中三篇是用snp array先找到了ALK amplification in
neuroblastoma，然后再对ALK测序，发现还有mutation。另一篇是用传统的lingage
analysis找到了ALK mutation in neuroblastoma。 |
|
l******u
发帖数: 936 | 35 not 大牛 or 牛, just some comments:
it is huge different between cells by FACS and in vivo.
(>30% genes differencially expression)
you could try tissue cryosection+LCM+RNA linear amplification+microarray/RNA
seq |
|
c******e
发帖数: 350 | 36 Sorry, but I don't have Chinese input here.
We have an old thermocycler (for PCR in capalliry) recommend a ramping rate
as high as 20C/sec.
When we ran the same PCR using a 96-well plate on regular thermocycler with
the regular ramping rate (2C/sec?), amplification was failed.
I wonder whether the failure is due to change in ramping rate? How does
ramping rate affect PCR?
Thanks a lot! |
|
c******e
发帖数: 350 | 37 Thanks.
But maybe I didn't explain well what happened.
We set up 2 reactions with identical reagents on 2 thermocyclers as below.
thermocycler Capillary 96-wells
Ramp rate (C/sec) 20 2
PCR volume 10 ul 10 ul
Amplification success fail
The reason we changed the ramp rate is that 2C/sec is recommended for the
96-well thermocylcer.
We didn't change reagents or Taq, be... 阅读全帖 |
|
f*******e
发帖数: 628 | 38 都有错误，所以 illumina 454 啥的都要事先 sample amplification。这个 nanopore
既然号称不需要 sample prep, 就需要解决单分子测序的 error 问题。当然如果
sample 本来就是 amplicon 就无所谓了。
哪个文章？他们网站上说是有两种 mode，其中一种是用 exonuclease 的。我理解那个
strand sequencing 不用 nuclease，但是具体怎么 把 double strand 弄成 single
strand，然后 single strand 通过了之后如何 recover 不是太明白。
我没以为是单细胞测序，所以才说 error 要通过 coverage 来解决。而 PacBio 的某
个解决 error 方法是循环的重复测某一个单分子，这一点 Nanopore 好像做不到，因
为我的理解是某个 strand 通过测序之后被重复测的几率很低很低。这个 error 的问
题，特别在 sample 本身很少的情况下，比如一个 finger stick 的血样，不能解决的
话就是个问题。 |
|
b****r
发帖数: 17995 | 39 谢谢
你读得比我更仔细,那个unzip double strand 的步骤确实没看到。
至于最后要多少个coverage，我没有算，你的描述里也没说出来，还是难以作出结论，
也许nanopore的coverage可以做到很高，这个还得花时间仔细算算。不过我觉得这个是
很难蒙混过关的，只要nanopore把数据拿出来，中学生也能算到很清楚，到底最后
coverage 多高，每个碱基精确性多高
你说的PCR even out error，我不太同意。你看看这个图
http://www.nature.com/nmeth/journal/v5/n12/fig_tab/nmeth.1270_F
如果我没搞错，illumina现在仍然是基于这个原理，我理解的PCR是左下最后一个蓝框
，不是右第三个黄框。前面这个amplification仍然是error prone的，特别是对于染色
体的很多区段，可能扩增效率很低，而且还有pcr的error问题和allelic dropoff的问
题（这些缺陷nanopore都应该没有）。实际上你去看illunima 的raw data，错误还是
相当多的，不是你... 阅读全帖 |
|
f*******e
发帖数: 628 | 40 我之前说的 amplification 的 error 就是指的在 bead 上面的 PCR，这个 error 是
在别的非 single molecule 方法里面可以 even out 的。之前 library prep 之类的
PCR，Nanopore 如果需要做，error 啊，bias 啊之类和其他方法相比没有劣势，大概
就是没必要用 adapter 吧。
需要多少 coverage, 我当然不知道，呵呵，每个系统可以自说自话，自己定义一个下
限，关键就是 customers 是不是接受了。还有就是用途不同，也许 4% 对有些人完全
不是问题。再加上 read length 长，这样 alignment 的 confidence 高，这里面如果
有因为 error 造成的 misalignment 也可以通过别的数据处理方法来弥补。 |
|
o*****r
发帖数: 39 | 41 The efficiency of single cell amplification of those two papers is
ridiculously high.
many people used the same kit, never got this good coverage.
The two paper will be under scrutiny. I have heard about bad comments about BGI.
We have to wait and see. |
|
l**********1
发帖数: 5204 | 42 PCR he noted. (possible)
Lutz S, Weber P, Focke M, Faltin B, Hoffmann J, Müller C, Mark D, Roth G, Munday P, Armes N, Piepenburg O, Zengerle R, von Stetten F
Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA).
(2010)
Lab Chip. 10(7):887-
full text link:
//www.imtek.de/content/pdf/public/2010/2010-04_microfluidic_lab-on-a-foil_for_nucleic.pdf
or
//www.bioss.uni-freiburg.de/cms/1644.html |
|
w****3
发帖数: 14 | 43 我也用过SMARTer™ RACE cDNA Amplification Kit (Clontech, 634923)，还行
，我花了3个月找到3个基因，在扩增不出来时你最好换一下不同条件下的total RNA,在
你的目标基因丰度越高时越容易扩增出来。 |
|
l**********1
发帖数: 5204 | 44 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 |
|
a****o
发帖数: 2 | 45 忘了补充两点
the PCR products for making qPCR standard also had smeary bands. I had tried
my best to minimized it by nested PCR with low concentration of DNA
template. But I assume certain unspecific amplifications still remained in
the qPCR standard, which could lead to smeary bands for the samples of qPCR
standard. It however does not explain the existence of smeary bands for the
tested samples.
Another thing it the Ct value is smaller than usual. |
|
c*******n
发帖数: 27 | 46 Check if your master mix contains the Rox dye. The machine can't determine
the baseline if there is no Rox dye, and you got no amplification curve. |
|
a*******o
发帖数: 16 | 47 最近用Taqman MGB probe做realtime也遇到问题，产物跑gel没问题，但是
amplification的曲线就是不对~ |
|
l**********1
发帖数: 5204 | 48 有师爷的 Stanislas Leibler在那里
师叔/伯的Uri Alon 在魏茨曼和哈佛两头站PI位 ps: Harvard is visiting PI.
and
Uri Alon and Naama Barkai both were his PD 15Y ago.
下边的师孙/孙女不成rising star 的天牛 不是得了癌症或其它致命的疾病中途弃权的
就是白犹里的white trash了吧? lol
Please refer:
Mechanisms of noise resistance in genetic oscillatiors
JM. Vilar, HY. Kueh, N. Barkai and S. Leibler
PNAS, 99, 5988-5992 (2002) | PDF
· Robust amplification in adaptive signal transduction networks
N. Barkai, U. Alon and S. Leibler
C.R. Acad. Sci. Ser. IV 2... 阅读全帖 |
|
a*****s
发帖数: 131 | 49 No-RT controls have no amplifications until 45 cycles... so I believe the
gene is expressed...
Thanks for more suggestions!
say |
|
n***w
发帖数: 2405 | 50 实验室人都走了。
神通广大的生物医学版，
96孔板做RT, 一排是4个样本，1个共同基因。
这些样本最后都有threshold cycle，但是两个样本没有melting curve （none）。
这到底是为啥呢？
肯定不是cdna的问题，因为其他的primers都work的不错。
觉得也不是primer的问题，因为其他样本有melting curve (偏偏就是我的KO没有。。
。。)
google了一下没找到符合的说法，基本都是有melting curve，没有amplification。。
。。
我是不是应该跑胶check一下。。
谢谢。 |
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