s******y
发帖数: 28562 | 1 一般方法里，我用过的是lipofectamine with Plus reagent 比较好，能达到
２０％而且细胞不容易死。
效率比较高的还有另外几个，但是细胞死得很厉害，你要是无所谓的话明天我给你
找找看是哪个厂家的。
病毒转染一般能达到７０％以上 （要注意细胞浓度，最好在５０％～７５％
confluence) |
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C****b
发帖数: 90 | 2 We have a RNAi screener who spent around 3 months to optimize the MEF
transfection condition. Finally she got 70~80% transfection efficiency.
Summary
Reagent: DharmaFECT
Cell: 30% confluent MEF instead of 70~80% confluent
Good luck! |
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M*****n
发帖数: 16729 | 3 Our friend and colleague Chi-Bin Chien died December 2, 2011, his battle
with cancer over. As many of you know, Chi-Bin defied his disease with
courage and optimism for over 12 years. During this period Chi-Bin
emerged as a leader in our field: He helped organize and lead our
international meetings, served as Director of the Zebrafish Neural
Development course at Woods Hole, cheerfully handed out new tools and
reagents his lab developed to help accelerate work with zebrafish,
brought us insights... 阅读全帖 |
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M*****n
发帖数: 16729 | 4 I heard he got cancer many years ago.
too sad I haven't had a chance to thank him for the reagents directly.
May his soul rest in peace! |
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n********k
发帖数: 2818 | 5 3k is big money for most of starting PI...some lab don't even spend that
much on reagents a month...except the past half y or so, I rarely burn more than 3k a month(mouse expense not included)..I have been the predominant money sucker over the ys in the lab... |
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c********y
发帖数: 25 | 6 buy a kit and read the manual, the library making procedure should be pretty
straightforward.
For e.g.
http://www.illumina.com/products/chip-seq_dna_sample_prep_kit.i
After you feel comfortable making chipseq library, you can figure out ways
of using homemade reagents instead of commercial ones, to save money.
BTW, you want to measure the DNA concentration using PicoGreen (or similar
stuff) but not Nanodrop. I made such a mistake before -_-! |
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s*****t
发帖数: 107 | 7 What spectometer you use for PicoGreen® reagent?
Thanks
pretty |
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y******8
发帖数: 1764 | 8 Illumina is way behind the competition. We need another serious player to
push ION on the improvement.
The good thing about ION is that reagents could be replaced by third party
products. The cost would be reduced to 20% of current price tag. In the
future, ION's profit could only come from two: chips and service. |
|
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t*d
发帖数: 1290 | 10 找到一点信息
The Ion Proton I Chip, which the firm said will be ideal for sequencing
exomes, will be available mid-2012. The Ion Proton II Chip, designed for
sequencing whole human genomes, will be available about six months later.
Lucier said that the cost of exome sequencing would be brought down to $500
with the new chip.
不知道这个是不是包括 chip 和 reagent 的价。是不是还包括了 exome capture 的
cost？现在 exome 的 cost 大概多少？
如果genome 比exome 大至少20倍吧，这样算，whole genome 需要 $10,000? |
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y******8
发帖数: 1764 | 11 Chip更新快还好，Kit更新快才是很麻烦。
不过可以推断出来，Ion的reagent没什么高深的东西在里面。等着VWR或者NEB出替代品
吧。
。。 |
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s******s
发帖数: 13035 | 12 ION的reagent应该很容易山寨，也是优势之一。人家主要就是卖Chip.
我前面提到了，这种东西我不信做不出reusable的，但是life肯定不干，
除非以后有新的盈利模式了 |
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n*********4
发帖数: 99 | 13 Illumina Introduces the HiSeq 2500
Speed and Performance Enhancements Pave the Way to Future Clinical Use
Illumina ILMN +2.61% today introduced the HiSeq(R) 2500, a next-generation
sequencing system that will enable researchers and clinicians to sequence an
entire genome in approximately 24 hours, "Genome in a Day". The HiSeq 2500
leverages the continued technology advancements from both the HiSeq 2000 and
MiSeq(TM) platforms.
"The ability to sequence an entire genome in a day with the same high... 阅读全帖 |
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l***y
发帖数: 638 | 14 1% efficiency is good enough, my cells can't even get 1%.
I have tried both electroporation and nucleofection for BAC, both work fine.
nucleofection is better and easier because reagents and parameters are
already optimized with the commerial kit.
350 |
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l***y
发帖数: 638 | 15 1% efficiency is good enough, my cells can't even get 1%.
I have tried both electroporation and nucleofection for BAC, both work fine.
nucleofection is better and easier because reagents and parameters are
already optimized with the commerial kit.
350 |
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n********k
发帖数: 2818 | 16 Hi, some of my thoughts from T's point of view---As it happened, I was in T'
s position for quite some time. I don't know your specific situations...However,
generally it is not a best position to be at for sure and I don't like it at
all, and I know many hate me in one way or another...but some one has to do
the dirty job in one way or anther and keep the lab functional...And many
times, it is the one who cares most or have the most interest at the stake..
.With my ys of experience on policing... 阅读全帖 |
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T****i
发帖数: 15191 | 17 I would use shared database, each student with different user name and
password.
There are tons of lab information managing systems (LIMS). Most of them only
work on PC. And a bad thing about them is that they were written by
programmers not biologists, so they are in general not intuitive, not
tailored to the needs of every biologist. Besides, usually you have to pay a
few hundred dollars a year. I think some may worth the money if your lab is
large enough. For smaller labs, it may be better to... 阅读全帖 |
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B****r
发帖数: 647 | 18 一个美国女生，人其实不坏，就是很喜欢竞争，很agressvie。最近我发现一个现象，
正在研究机理，她就派她的undergrad做我现在正在做的东西，说如果这个undergrad做
出来了，她和这个undergrad就可以继续研究下去blahblah，我很生气跟她说不可以，
因为这是我的project。开始我还以为是老板的主意，后来她说是她的主意。。
是我太overreacting了还是这种行为确实属于不道德啊？她一向都很喜欢竞争，比我低
两个年级，但是总想比我做得好啊还想跟我一起毕业，我都无语了。
有经验的前辈说说这种情况咋办吧。我们lab其实气氛很好的，大家都互相学习的，还
是我当年train的她。现在她成天拿我的reagent和antibody用都不告诉我，我因为这个
都跟她说过一次了。她是第二年phd了，我是第四年。
谢谢！ |
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w******y
发帖数: 2504 | 19 谢谢回复。难道没有什么REAGENT来诱导细胞衰老么？ |
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l**********1
发帖数: 5204 | 20 RE: 德艺双馨的楼主
please go to
//www.creativebiomart.net/description_10243_28.htm
You Position: Home >> Antibody >> Polyclonal Antibody >> Rabbit PAb >> Anti-Cdh5 PAb
Anti-Cdh5 PAb
Cat. No.
CPB-133RM
>
Immunogen
Recombinant mouse CDH5 protein.
Antibody Type
Rabbit Polyclonal Antibody.
Ig Type
Rabbit IgG.
Formulation
0.2 μm filtered solution in PBS with 5% trehalose
Preparation
Produced in rabbits immunized with purified, human cell-derived, recombinant mouse CDH5 extracellula... 阅读全帖 |
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a*****x
发帖数: 901 | 21 Totally agree. The other thing is that whether you can get all the reagents/
equipments in China |
|
l**********1
发帖数: 5204 | 22 Sure:
Post- CoCom:
>In addition, those entries and sub-entries listed in
Supplement No. 1 to this Part 771 are eligible for export under this
general license. However, this general license is not available for
items that are also subject to missile technology (MT), nuclear
nonproliferation (NP), or foreign policy (FP) controls to the recipient
country.
(c) Eligible consignees. This general license is available only for
exports to civil end-users for civil end-uses. Exports under this
general... 阅读全帖 |
|
s******y
发帖数: 28562 | 23 I think nowaday many Chinese university/institute have better equipments
than their peers in US.
reagents/ |
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l**********1
发帖数: 5204 | 24 try
Human Phospho-MAPK Array kit
(R&D Systems, Minneapolis, MN).
link:
//www.biocompare.com/ProductDetails/3015549/Proteome-Profiler-Human-Phospho-
MAPK-Array-Kit-NEW.html
cited from 2011 paper:
Activation of p38 mitogen-activated protein kinase by norepinephrine in T-
lineage cells.
Immunology. 2011 Feb;132(2):197-
link:
//www.ncbi.nlm.nih.gov/pubmed/21039464
>Phospho-MAPK protein array
>Following exposure to NE (150 lM, 15 min), total cellular
>protein was obtained using the reagents and proto... 阅读全帖 |
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T****i
发帖数: 15191 | 25 我不知道别人是否重复过，但我自己都能重复的才会发。不敢说100％可重复（因为有
strain background, reagents, 等等细节问题），但key data都可以重复。Key data
必须robust。
我追求的是consistent reproducibility。如果一个实验我做几次结果不一致，我会用
其他手段再验证。
其实很多时候结果和自己想的不一样恰好是发现新现象的契机。 |
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s******s
发帖数: 13035 | 26 这个看community是不是健康了。像做果蝇的，大多数只要publish的突变
都能要到，顺带reagent比如Ab什么的也比较好要。做老鼠的就比较难的，
做cancer的更难，如果是中国lab或者美国这里中国人开的lab，基本上就
是没戏。所以做fly genetics就很难造假 |
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l**********1
发帖数: 5204 | 27 Sure?
你觉得你做的KO Mice 研究(2nd round PD) paper results parts
有几成(or how many %) 是能被别lab重复的？
新一代坑王的
Key data 必须robust=95% 能被别lab重复的信心吧 啊?
---
[回复]
[ 3 ]
发信人: Tianzi (tt), 信区: Biology
标 题: Re: 看了”大部分cancer research结果都不能重复“一贴想起的
发信站: BBS 未名空间站 (Sat Apr 7 08:59:33 2012, 美东)
我不知道别人是否重复过，但我自己都能重复的才会发。不敢说100％可重复（因为有
strain background, reagents, 等等细节问题），但key data都可以重复。Key data
必须robust。
我追求的是consistent reproducibility。如果一个实验我做几次结果不一致，我会用
其他手段再验证。
其实很多时候结果和自己想的不一样恰好是发现新现象的契机。 |
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n********k
发帖数: 2818 | 28 For my main project, at SFN when we presented the study, quite a few asked
me where we had sent--science or nature?...In reality, we started with Cell
(I personally know it is not cell level but we tried anyway:)))...and then
went down the list---luckily/unluckily, it never made through the editors in
any of the top journals...so within 2-3 months, it went to the journal that
eventually accepted the MS and the entirely reviewing process took about 3
months...It is fair to say the study should h... 阅读全帖 |
|
n********k
发帖数: 2818 | 29 yeah, Amax lacks flexibilities, all you can do is to following the protocols
...but some say it has better efficiency..but if you can get 50% for B and T
cells, I would say that's very good...what about the survival rate? Amax
survival rate is horrible...although the efficiency is very good for our
cells...BTW, how much the reagents cost?
more |
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d****d
发帖数: 214 | 30 Some insightful comments on the article:
JJ Bangor, ME
In years past, Nature, Science and the Cell Press Journals would devote
maximal energy to make sure scientific accuracy was maintained for all
articles that were published on their pages. Sadly, this is no longer the
case. In fact, a journal's impact factor is only helped by bad papers that
attract a lot of attention and are later found out to be fraudulent or
simply wrong. This is the case simply because these papers continue to be
cited... 阅读全帖 |
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n***w
发帖数: 2405 | 31 My mentor also discussed this article with me today, saying that whenever I
publish a paper, I need to put all raw data [all triplicate experiments, the
whole blot (not just a cut band), etc] in one file so he can track. lol
Also we think that those reagent companies should be under federal
regulation, as a majority of grant money goes to antibodies and all such,
which may not work at all. This is very irritating... |
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f*******e
发帖数: 628 | 32 这个问题真是不好答。
background 总会存在， 不影响 signal 的辨别的话，不用去除，直接处理 image 就
好了。
实在是要 physically 去除 background，就得知道是哪里产生的，sample，substrate
，reagent, buffer,microscope 都有可能，那就要对症下药了。 |
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c**********e
发帖数: 230 | 33 millipore有个non-protein的blocking reagent，可以让你western完了以后再用一般
的染胶的方法染膜，效果还可以 |
|
n********k
发帖数: 2818 | 34 Never work with invitrogen cloning kit since I used strategen ones...so no
idea about the comparison
BTW, to save, one could cut down the reaction volume to a half or even 1/4
with all the reagents, including the competent cells(20-25ul is very safe,
10-15ul is fine), so with the 20 reaction kit, I can get at least 40-80
reactions...there was time I need to do 20-50 cloning or something like that
in a week... |
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C****c
发帖数: 1964 | 35 在美国PhD第五年，刚刚答辩完，碰到一个极品系主任，很愤怒，可是不知道怎么办。
事情是这样的，我老板是一个华裔女PI，五年前recruited到中部排名160左右的生物系
做assistant professor。中间不知道什么事情得罪了系主任（我老板到现在都不知道
），系主任以我们老板没有paper为由，overturned系里老师投票的结果，不给我老板
最后一年的reappointment（注意不是tenure，是拿到tenure之前最后一年的
reappointment）。我老板一级一级上告，在college投票结果通过但又被dean
overturned了。后来我老板觉得上面已经和系主任达成某种默契，最后放弃。我们做发
育，出东西比较慢一点，现在还一篇paper都没发，但是有6篇paper在写准备投出去，
有几篇应该能投不错的杂志。而且我们申请经费没有任何问题。现在我老板在我们学校
的另外一个系找了个associated professor的职位，我们系主任开始丧心病狂了，要求
我们实验室6月21号左右搬出去，给系里的其它所有老师发了一封email（除了我们实验
室），说我们实... 阅读全帖 |
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y******8
发帖数: 1764 | 36 Overall, I would agree with some replies. Engineering can be big, but
science
should stay small. High efficiency could be at the high cost of creativity.
In general, ideas are cheap in biomedical research, because there are
hundreds of thousands times of more ideas than data. The only arguable
exception might be sequencing. Still, I personally think there are far less
data than desired for a theoretic biologist (not a computational biologist).
There are tons of cores, and service companies. In ... 阅读全帖 |
|
n***w
发帖数: 2405 | 37 读paper都是用zymograph来看MMP的表达，或者qPCR.
可以用染色来看在组织上的location吗？还是目前reagent不允许？
Thank you! |
|
s******y
发帖数: 28562 | 38 啊？那也太低了！！！！
我也是用这个细胞系，最少也能搞个10%。
在我的经验里，lipofectamine + Plus reagent 最好用，能够达到20%。
关键就是要比厂家porotocol 里面说的plasmid用量加倍 |
|
w***a
发帖数: 4361 | 39 HHMI的elife还是以前的老思路，没太可能冲击CNS的地位。
最多也就是另一个plos biology，没有新意。
像HHMI，welcome trust这种不差钱又有一定公信力的机构，完全应该自己建个中心实
验室，
雇几百个PhD。
凡是投elife杂志的，最后一遍重复试验要去HHMI的core lab做。要么提供reagent让
HHMI的wsn phd来重复key data，要么自己派人过去在监督下重复。
这么搞的话，elife可以保证所有publish出来的data都能够重复，那不出两年肯定完胜
CNS。
nice
nothing
leading
curiosity |
|
p*****m
发帖数: 7030 | 40 老板署名做通讯作者 在实验科学我不认为有太不对的 毕竟学生博后可以很快走人 以
后的读者想要reagent想要reprint找谁去？
但是把通讯作者等同于提出想法贡献智慧的人，然后大部分的credit都给通讯作者，这
个我是坚决反对的。
奖。 |
|
n******7
发帖数: 12463 | 41 Resisting Arsenic
The discovery of a bacterium living in the extreme conditions of Mono Lake,
California, created a major controversy because it was claimed to be able to
grow solely on arsenic and could substitute arsenate for phosphate in its
key macromolecules, including DNA. Working with the same Halomonas spp.
bacterium, known as GFAJ-1, and ultrapure reagents, Erb et al. (p. 467,
published online 8 July) found that the bacterium needed a low level of
phosphate (1.6 μM) to grow at all. Rath... 阅读全帖 |
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b******s
发帖数: 5365 | 42 用的抗体和isotype都是和flow一样的.做confocal 时先用4%PFA fix然后用0.1%
triton-X100 permeablize.应该不是通透性的问题,因为我同时染了另一个细胞内蛋白,
那个的染色信号就很好.
请问你用什么做block FcR?你说一起做,是不是指把block FcR的 reagent 和二抗血清
混合一起孵?谢谢!
cofocal
内。 |
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E*********s
发帖数: 93 | 43 我用oligo synthsize 2nd strand. 但是合成后如何才能区别dsDNA 和ssDNA？ 最好是
哪个gel staining reagent 可以直接通过跑gel 就可以知道。谢谢。 |
|
s*****g
发帖数: 7857 | 44 电转shRNA / siRNA 不行吗?
哪个什么GENE6(似乎是这名,好久不用了)可以吗? |
|
g*********5
发帖数: 2533 | 45 要降低siRNA浓度。
试过lipofectamineRNAimax 1-10nM了吗? |
|
|
S*********u
发帖数: 980 | 47 我用过QIAGEN hiperfect 效果很好，毒性很低，不过转的是cancer cell |
|
m*********7
发帖数: 5207 | 48 Have you tried nucleofection? |
|
b**********8
发帖数: 349 | 49 DharmaFECT 4 works well all the time in our lab. We regularly handle with
PLC5,Hep3B,HepG2 and Huh7 cells. |
|
|
