R*****w
发帖数: 447 | 1 直接注射是可以的，只是转化效率很低。至少肌肉注射ＤＮＡ可以表达目的基因、刺激
机体产生抗体，但要多次大量注射才行（50ug/20gmouse/dose,３－４次）
in vivo 用transfection reagent 效果不明显 |
|
b******d
发帖数: 149 | 2
其是
对而
I think that 非学术因素的干扰 in China is very much much worse.
Here are a few disadvantages for NIBS in China:
1) Quality peers outside NIBS are much much worse in China, this also
reflects on seminar qualities, conference and meetings.
2) Collaborations between NIBS scientists (PIs/postdoc/students) and
other international scientists are not as easy as PIs overseas. This is
true for reagent purchases and requests too.
3) I would handicap the political environment in China against NIBS as
well,... 阅读全帖 |
|
H*****e
发帖数: 120 | 3 Finally!
Amy is a star for long time. But almost like a joke although she and her
supporters fighted hard. Many years ago, she wrote a Cell review saying no
transdifferentiation. A few weeks later, there were 2 science paper did
transdifferentiation. When Cell launched Stem Cell, she was in the editor
board then she was elected for the senior post position in stem cell
association. How many AP could get this kind of treatment? I guess she
must make a big mistake in it this time.
Another ri... 阅读全帖 |
|
m*********7
发帖数: 5207 | 4 想从某collaborator那里拿到一些质粒和其他reagents，可是人家推三阻四不愿意给。
我老板猜测，那个教授担心我们会成为他的竞争者。怎么才能消除他的这种疑虑呢？因
为不想把实验计划跟他说的太详细，又不知道怎么说才能有说服力。 |
|
s******y
发帖数: 28562 | 5 Agree. Mycoplasma is usually derived from reagent such as serum, not from
human, unless you have an active mycoplamsa infection in your lung and
happen to cough into the culture hood.
project |
|
k****y
发帖数: 423 | 6 不用过分自责，我怀疑你们实验室的细胞本来就有myco污染，只不过你用的细胞比较敏
感，所以raise the issue,不然说不定都发现不了。很多细胞特别是贴壁细胞的growth
/proliferation 在myco污染之后都没有影响的。再者如果细胞比较难获得，可以不用
扔掉，用mycoplasma removal reagent，比如plasmocin就可以，一般2-3个星期就可以
除掉。以后实验室常备mycoplasma detection kit，隔段时间测一次就行。 |
|
x****z
发帖数: 3473 | 7 mycroplasma contamination is not likely to be airborn, not likely due to
your technique.
Must from some reagent contamination. It's not a big deal. Don't worry too
much. |
|
y******8
发帖数: 1764 | 8 One sample from google. Good luck!
Dear PLoS Biology editor:
I would like to submit a presubmission inquiry regarding suitability of our
manuscript, "Subversion of cellular autophagy pathway by RNA viruses" by
William T. Jackson, Thomas H. Giddings Jr., Sara Mulinyawe, Ron Kopito and
Karla Kirkegaard for publication in PLoS Biology. Cellular autophagy is a
field that is just entering a period of rapid discovery, because the
mammalian homologs for the many yeast genes that affect autophagy have v... 阅读全帖 |
|
b******d
发帖数: 149 | 9
gave
the
Putting graduate student/postdoc as co-corresponding author also release
the burden of future reagent requests and communication stuff. PI could
just let graduate student/postdoc take care of it in the future.
Corresponding authorship is NOT for nothing :) |
|
p*****m
发帖数: 7030 | 10 不是做鱼的 但是对鱼很感兴趣 抛砖引玉说几句吧 期待内行指点
首先 鱼这东西确实是稍微基础了点（比起rodent models），在NIH系统申请钱的难度
应该可以归入worm, fly这些最基础的模式生物里面 尽管人家是脊椎动物。。没办法
rodent people觉得只有他们才是搞human related的。公司里面，尽管也有公司试图把
鱼做起来（主要是biosafety/toxicity，也有检测环境质量比如雌激素什么的，也有真
的想拿fish做disease model/drug screen的），但是总的来说fish离一个成熟应用的
系统还差得远 一般的疾病/药物研究，如果不是直接从in vitro跳到clinical的，一般
还是喜欢大动物，rat/rabbit/dog，必须genetics那就mouse。
不过话说回来，我自己对fish来做基础的生物研究还是很看好的。。一方面看得见这个
优势太大了，一方面small molecule screen可以没事拿来做做chemical genetics啥的
（虽然真的要找target也不太靠谱吧 呵呵），或者代替繁琐的遗传操作... 阅读全帖 |
|
f**u
发帖数: 346 | 11 呵呵，做果蝇的团结开放绝对可以算得上一大美谈，
在果蝇领域问别人要reagents什么的都是过两天就给你寄过来。
听说做老鼠的就。。。 |
|
M*****n
发帖数: 16729 | 12 how long is your PCR product?
what is your reaction volume?
My suspicion is you used too much template (very likely) or too little
template, given that all the other reagents (such as primers etc. ) are OK.
most likely you need reduce your template. |
|
B*H
发帖数: 2158 | 13 Qiagen的我也用过，是很不错的。就是blocking reagent是他们自己秘方，奶粉不行，
降低灵敏度。300多一个kit，还是蛮耐用的，抗体稀释一次可以用好几次呢，－20度保
存一两周没问题。 |
|
w***a
发帖数: 4361 | 14 RXi那个东西貌似原理没公开，俺们lab有人在细胞系里用过，说是效果跟用liposome t
ransfection差不多，但是不需要transfect reagent。
但是it的siRNA并没有overcome delivery的问题，直接iv injection，还是会降解，我
看也没多大进步。 |
|
S*******m
发帖数: 34 | 15 点击每一个Retraction的链接就行了.注意这一句:
Dr. Suresh Radhakrishnan, who was involved in or had access to all work on
this subject, was found in a formal institutional investigation to have
engaged in scientific misconduct in unpublished experiments by
manipulating another investigator's experiment involving the B7-DCXAb
reagent. |
|
l**********n
发帖数: 240 | 16 三哥就是猛！
不过不明白，好像有几篇的作者中没有他的名字。
难道是试验中用了B7-DCXAb
reagent which has been manipulated by this guy? |
|
z*h
发帖数: 773 | 17 So you protect yourself at the beginning. When I had a revolutionized idea (
I think that it deserved Nobel Prize surely) several years ago, I did not
share the idea with anyone, even with my outside collaborators because I
need borrow instrument and obtain key reagents from them. No presentation.
Only filed patent disclosure. After the paper came out, we started
presenting this breakthrough. The bad thing is that without initial
marketing, it cannot be published in top journals. But gold will... 阅读全帖 |
|
n********b
发帖数: 27 | 18 There is a Research Associate II position opening in a Genomics-Microarray
Core in a Cancer Center in southern CA-Los Angeles Area. Please send your
resume/CV to o****[email protected], if you are interested
in it. Thanks.
See job descriptions:
Job Descriptions
Research Associate II
Functional Genomics Core
1. Perform bench work related to research projects & services using
different genomic technologies including Affymetrix GeneChip, Agilent, Roche
NimbleGen and Illumina’s HiScanSQ microarray platfo... 阅读全帖 |
|
C*******e
发帖数: 4348 | 19 呵呵
我觉得比较重要的是第一步trizol
（或者别的phenol based reagent不管每个公司起的什么名字）
和起始材料之间的比例比较重要
另外我自己做过对比
phenol based方法不走colum比走column产量多出来至少5倍以上
对于起始材料足够多，或者下游实验对于产量没有太大要求的实验来说自然column方便
省事
不过有时候还是需要用old fashioned method啊 |
|
w******e
发帖数: 1187 | 20 in theory protein应该也可以被transfect吧？也许可以用来做些instant
manipulation of cells w/o long term disturbance.
有没有这方面的文献？多谢！ |
|
z***x
发帖数: 80 | 21 I have no experience of isolating RNA from cartilage but need to do it soon.
Cartilage contains a lot of proteoglycans and glycosaminoglycans (GAGs). I
first homogenized cartilage into powders by freeze-milling. I then tried
guanidine and phenol to dissolve the cartilage powder but it was not quite
efficient. Most of the 100mg of cartilage powder was not dissolved in 2 ml
guanidine/phenol (Qiagen tissue lysis reagent).
The other question is that RNA and GAGs are both negatively charged polymers
... 阅读全帖 |
|
b******n
发帖数: 4225 | 22 我们学校很快有ion torrent的测序服务了
学校测序中心的人说不算人工，成本就$500/run
$250 chip, $250 reagent
100M/run
估计加上人工大概$700左右吧
最重要的还是后面的reads->contig assembly |
|
K***n
发帖数: 131 | 23 Where can I find the agents from whom reagents for biomedical research can
be purchased and re-sell to China? Thank for your information. |
|
z*******a
发帖数: 175 | 24 在生物版受益良多
此前发文多为技术求助，谢大侠们回应帮助
潜水专家，少有评论。2011将暨之时，借Linda Buck撤稿事件聊发感言，籍此存照自勉
1，定性结果（有或无）必须自己重复两次以上，定量结果based on三次以上
replicates。注明所用条件，包括动物
strain，source， reagent的source
2，大牛忽悠的是interpretation，model 和 conclusion
3，interpretation 和 conclusion 可以错，data错了就别奢望他人大肚能容。自己发
现interpretation 和 conclusion 有
错后，及早发篇新文（IF may less than 1）更正之
4，文章可少发，信誉不可轻。类邹之赌徒心理决不可存
5，诚实为人，勤精为业，路总会越走越宽，除非walmart模式在academia成真。严防急
功近利，千万别轻生，留的青山
在吧
6，买房租房时，人都说中国人有信用。诚望有一天，人人都说中国人第一作的paper可
信没商量。被说成cheap foreign
labour 真是好没面子，诚望x... 阅读全帖 |
|
m***T
发帖数: 11058 | 25 二代技术靠什么赚钱？机器本身只是一部分，另外的可持续性的营收还是在于reagents
。SOLiD的一个优势正在于此，invitrogen和ambion分别提供DNA和RNA的kits，这点儿
ILMN是比不上的。另外，由于是dibase，所以accurate rate会略高些。ILMN的优势在
于市场份额（处于dominate的地位）以及简单的workflow。至于454的位置则比较尴尬
，throughput比不上前两家，read length又比不上三代技术，所以市场应该会进一步
萎缩。
三代技术目前看还不够成熟，以领先的PacBio为代表，error rate高及throughput低的
问题都还没有得到有效的解决，而且这些不是短期内就能解决得了的。我个人感觉
PacBio野心太大，最近一年来扩展的步伐相当大，招了不少可招可不招的人，对于一个
产品没有上市，完全靠烧钱过日子的startup来说它的步子太大了点儿。它的这种
marketing strategy比较risky，如果成功可以一下领先对手不少，一旦结果不好，甚
至有可能会重蹈helicos的覆辙。
严格意义上讲Comp... 阅读全帖 |
|
j****x
发帖数: 1704 | 26 论挣流水钱，目前确实没有哪家能和Life Tec这头航母比，但是这块的门槛相对较低，
试剂的利润率基本也都是摆明的，被“山寨”也是摆明的。对ABI而言是优势，但是
目前完全不足以挽回SOLiD的全面劣势。
SOLiD是个很奇怪的东西，技术层面理论上说它算是2代中最突出的，但一方面因为
出来的晚，更重要的是，ABI的相关配套很差，workflow非常不友好，下游的数据分析不
兼容，自己的算法一直也没跟上。最终的结果就是没人用，很多地方买了也是放起来。
2代中就数它悲剧，未来个人也不看好。
ILMN不说了，一个公司从追赶者能做到这个份上，不容易了。无论是它的array还是
sequencer或者其他的产品。它的存在是二代技术能如此迅速普及，花费下降如此之快
的最重要原因之一。
454未来相信会做细分市场，比如推出的GS Junior，面向临床的小型化平台化对roche
而言是不错的思路，毕竟诊断这块是roche的强项，利润也不是普通实验室试剂所能
比得，门槛也高的多。份额会萎缩，但是收益未必。
三代技术目前论之尚早，抛开还没有经历过任何商业化的实战考验不论，技术上离实用
还太远，只能作为概... 阅读全帖 |
|
m***T
发帖数: 11058 | 27 想看具体的individual文章们，请follow最下面的link.
NEW YORK (GenomeWeb News) – In a very busy year for news in the 'omics and
molecular diagnostics industries, an article about an acquisition topped the
list of the most-read stories for the year on GenomeWeb Daily News, while
restructurings, lawsuits, and one firm's IPO plans made it into the top 10.
Here are the top 10 for the year:
1) Life Technologies CEO Defends Price for Ion Torrent, Dismisses Illumina's
PCR System
2) Roche Restructuring Plan Includes 4,8... 阅读全帖 |
|
m**********d
发帖数: 137 | 28 问了一圈人，可能是pPCR machine跟reagent不compatible
实验室的qPCR仪是ABI 7300，订的是bio-rad的SYBR,据说要想用这个机器需要另加ROX
，或者换到ABI7900机器，或者从ABI订SYBR green(has ROX in it)
dissociation |
|
c******r
发帖数: 3778 | 29 嗨，如果这个问题好解决。
7300用ROX做internal reference。bio-rad没有ROX。不过没关系，7300可以调设置，
直接把ROX的reading去掉就可以了。你看看manual，自己设个新的program，不用ROX就
可以了，不用换reagent。
ROX |
|
m******5
发帖数: 1383 | 30 如题，现在很多Protocol都写磁珠了，不知是一定要用还是可以像做co-ip那样用一般
珠子 |
|
|
p*****m
发帖数: 7030 | 32 这是个问题 说大点就是fish有点不尴不尬的，如果说做保守的东西还不如fly/worm又快
又便宜 reagent也发达；如果说一定要接近人，那fish和mammal还是差的挺多的，就连
frog都不如。说了20多年还是坐定一个所谓的研究vertebrate发育的model来用，我一个
门外汉的感觉就是有点为了搞个model system而model system的意思。
我感觉现在可以慢慢的用fish做一点本来设计之外的东东了 说不定可以更好地利用fis
h的优点。比如说，体外受精这个好处是不是可以用来系统的做epigenetic control,甚
至做reprograming的东西（如果frog不方便的话）？还有，larvae早期脑袋还是透明的
，用来搞很fancy的neural circuitry study会不会很爽？用来做neurodegeneration/r
egeneration会不会很方便？更不要说drug screen了，如果能想出好的assay来潜力非常
大啊，最近几个用fish做的发育之外的screen都很有意思，一个sleep的，一个optomot
or re... 阅读全帖 |
|
b*********l
发帖数: 490 | 33 I am thinking another issue. During the process of zebrafish like fixation-
dehydration-rehydration, all of the MeOH+PBT solution should be with DEPC
treated water or what? Is it possible that the mRNAs in zebrafish have been
deteriated before the hybridizaion? I am not sure how bad the
invivo mRNA will be if I don't use RNase free reagents. The thing is I
couldn't see any difference between negative control and the real samples.
Thank you for any suggestions. |
|
s******s
发帖数: 13035 | 34 NGS is cheaper if you don't consider the price of the machine.
I believe a lane of flow cell is about $0.5k, and home-made
reagent is very cheap. |
|
w******y
发帖数: 8040 | 35 home made reagents?
Can they work ? |
|
W****C
发帖数: 1937 | 36 ｔｒａｎｓｆｅｃｔｉｏｎ 没ｗｏｒｋ。。。不行就换transfection reagent, 比如
ｆｕｌｌｇｅｎｅ |
|
t****t
发帖数: 610 | 37 Thanks. It's Methylation Specific-HRM. I'm studying the same CpG islands,
so primers are all the same.
Primers do flank CpG sites.My primers work.
Even I have PCR product contamination, how it is possible to get a Tm lower
than unmethylated control? I have PCR controls, which are already converted
standard DNA (methy and unmethy). They were fine with the right Tms.
Also, although I'm using the same reagents (kit, buffer,...), last week
there was once that my results were fine. Really got confus... 阅读全帖 |
|
g*********d
发帖数: 233 | 38 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 |
|
c********l
发帖数: 244 | 39 Of course you can do it. Just follow the protocol for the transfection
reagent that you are going to use. I personally used hiperfect and
oligofectamine. |
|
n********k
发帖数: 2818 | 40 仅有几
个克隆，送过去测序，要做的8个构建株里面只有一个对的，其他基本上都是在Bgl2那
一段的引物地方要么有突变要么就是缺失。
well, could you start with these wrong ones now and just do some mutagenesis
, like you did with dpn1 ones...it would be much easier if it were due to
digestion/ligation etc...but it won't solve the problem if your fragments
are toxic or have too many repeats etc...It is a bit strange that it only
happen to your primer region in all the constructions though...if it is
toxic or due to recombination, it could happen to any regions or... 阅读全帖 |
|
n********k
发帖数: 2818 | 41 谢谢你的详细意见。首先要说，我做分子克隆的经验不是很多。其次，基本不会不改变
条件反复重复。
1 当初选用pQE8也是没有办法，因为pQE9我们实验室没有（pQE8和9的区别就是8在酶切
位点之间只有一个碱基，而9要长得多）。最开始的时候没有意识如此近的酶切位点很
难切下来，最起码同时酶切不可能切下来。但是只尝试过一次顺序酶切不行就没有再尝
试用pQE8了，我不记得是先切BamH1还是Hind3，后面尝试插一段片段在2个酶切位点之
间再切。
Nice alternative, shall solve the problem in this case...but I would just do
blunt ligation, nowadays, the fast ligation kits from different companies
such as Roche, takara, don't really care about whether it is sticky or blunt
ends, it works just as well...BTW, any one else ... 阅读全帖 |
|
h*******o
发帖数: 4884 | 42 I don't recommend H2O2, as the reagent itself also elicits DCF fluorescent
signal. Then it will be too hard to distinguish between ROS generated by the cells and ROS attributed to H2O2.
Also, you might want to consider MtSOX for your assay. |
|
F*****e
发帖数: 182 | 43 准备从wildtype和mutant的老鼠里取mouse tissue，提取total RNA后做mRNA seqencing。
在犹豫是用Trizol还是Qiagen RNeasy Protect Mini Kit，因为需要先取胚胎，然后
PCR鉴定genotype，之后才能决定需要做哪个，因此最好能将tissue保存一段时间。
Qiagen的kit里面有一个RNAlater RNA Stabilization Reagent，号称tissue放在里面
可以4度保存4周而RNA不被降解，真的这么安全么？有没有用过这个kit的朋友。
另外mRNA sequencing给的结果到底有多可信？定量分析怎么样？多谢。 |
|
s******r
发帖数: 2876 | 44 提RNA的kit不贵，你完全可以全部都提，
等genotype结果出来吧不要的扔掉。
准备从wildtype和mutant的老鼠里取mouse tissue，提取total RNA后做mRNA
seqencing。
在犹豫是用Trizol还是Qiagen RNeasy Protect Mini Kit，因为需要先取胚胎，然后
PCR鉴定genotype，之后才能决定需要做哪个，因此最好能将tissue保存一段时间。
Qiagen的kit里面有一个RNAlater RNA Stabilization Reagent，号称tissue放在里面
可以4度保存4周而RNA不被降解，真的这么安全么？有没有用过这个kit的朋友。
另外mRNA sequencing给的结果到底有多可信？定量分析怎么样？多谢。 |
|
h****n
发帖数: 2552 | 45 Fixation reagent is no use to help cells attch on a plate. I suggest coating
your
plate with poly-lysine
is |
|
l******a
发帖数: 3339 | 46 You can use Detergent compatible BCA assay from Biorad. Or Ellman's
reagent for thiol containing protein.
bound
phospholipid
how |
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c***c
发帖数: 113 | 47 Thanks.
I read something about Pyro sequencing online, it seems to do pyro
sequencing, special instrument and reagents need to be bought. Could you
please tell me what do you have for pyro sequencing assay and how did you
design the primers? Currently we just want to check one gene of interest, we
will not do DNA methylation assay routinely. |
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f*******e
发帖数: 628 | 48 bioanalyzer 主要是 kit 很费钱。如果 lab 用得不多，其实没有必要买。
用过的 chip 一次能跑 10 个样品，一个 kit 里面有好几个 chip,然后一套 reagents。 |
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W*******a
发帖数: 1769 | 49 做high throughput seq的，bioanalyzer还是必须的
reagents。 |
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g****0
发帖数: 425 | 50 an antibody good for western-blotting is not necessary a good one for IHC or
IF. You can try it on cell culture first. How about IP with KO as control?
It is definitely a way to go try different antigen retrieval methods.
It is also useful to try different blocking reagents. |
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