y**u
发帖数: 7459 | 1 rt. 或者其他什么primary cell？
我知道有用lipofectamine transfect primary neuron的。有没有其他经验可以借鉴的
？just curious. Thank you! |
|
m******g
发帖数: 69 | 2 我们买了一种小的LNA,用了沉默一种miRNA,可是用什么样的条件才能transfect LNA到
cultured cell里面，来达到沉默miRNA. 谁做个相关的实验，有paper和protocol吗，
我是本科生，老板让我做这个，没什么头绪。 |
|
g*********5
发帖数: 2533 | 3 transfected them in 6 wells and after 24 hour trypsinzed cells , count, seed
them in chambers. |
|
D***a
发帖数: 516 | 4 希望找一个能像HEK293一样容易转，但细胞个头要大，能在玻片上铺得开的。
另外问个问题，transfection效率跟细胞的什么性质有关？为什么有的细胞好转，有的
难转？ |
|
s******y
发帖数: 28562 | 5 transfection 主要和两个因素有关：
１。膜的活跃程度：喜欢ruffling 的细胞容易转
２。细胞周期状态。频繁处于复制周期的细胞就容易转。
个头大的细胞挺多，但是既好转又容易铺开的。。。我还真不知道。 |
|
c****g
发帖数: 93 | 6 我们的经验表明如果胞内cholesterol比较多的情况下，膜上一般也比较多，这时候很
不容易转进去。所以我就想认为deplet cholesterol之后在做transfection不知道会好
些不。我没有test这个。 |
|
a***u
发帖数: 155 | 7 我要用lipfectamine转siRNA，hela 细胞，是在20 cm的大dish里直接做好呢，还是先
在 10 cm的dish做transfection，然后移到 20 cm的大盘子里面长两天？我很讨厌用
lipfectamine，太毒了，但老板不愿意另买试剂了
谢谢！ |
|
y*********u
发帖数: 183 | 8 Be more specific?
你是指stable transfection? |
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a******a
发帖数: 313 | 9 请推荐 siRNA Transfection reagent for hepatocellular cells that does not
activate autophagy.
试过Lipofectamine 2000，LC3的表达会改变。也试过DharmaFECT 4，没有转染成功。
多谢各位！ |
|
b*******n
发帖数: 605 | 10 有人说Effectene Transfection Reagent (Qiagen)好，
有人说Lipofectaminehao好，
有人说。。。
做stable cell line,到底哪个比较好？
谢谢！ |
|
A******y
发帖数: 2041 | 11 If they are leukemia cell line, normal transfection reagent will not work. |
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h******y
发帖数: 351 | 12 Thank you.
Do you know why transfection reagent(s) are toxic to cells? |
|
h******y
发帖数: 351 | 13 没看懂。这怎么能和transfection 试剂的毒性联系起来呢？ |
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l**********1
发帖数: 5204 | 14 楼主每隔24 hrs transfection 外来shRNA/miRNA 可以保证对宿主细胞 oxidative
stress/heat effect/ injury的
是(- -) 吗?
如果楼主确信 那可不考虑分子伴侣 hsp families 的作用
如果需考虑可能有(- +) or even (+ +) effect of oxidative stress/heat
effect/ injury caused hsp90/70 actions,
pls refer: Mammalian 肾细胞的hsp90 one paper:
by Harrison EM et al. (2008)
Heat shock protein 90-binding agents protect renal cells from oxidative
stress and reduce kidney ischemia-reperfusion injury.
Am J Physiol Renal Physiol. 295: F397-405.
web link:
HTTP: /... 阅读全帖 |
|
|
m********o
发帖数: 13 | 16 想在Hela转一个GFP tag的蛋白，试过lipofectamine和xtremegene效果都不好，基本上
看不到荧光。同样的条件在293细胞里转得还不错。求助哪位有经验的有没有别的
transfection reagent推荐，或者有没有什么tips？谢谢~ |
|
d*********e
发帖数: 876 | 17 效果如何？打算试试ATCC的 MEF (CF-1) (ATCC® SCRC-1040™)。
或者有没有推荐的 mouse cell line 作为 transfection host?
之前用 NIH-3T3 做转染,但是很容易就长出 spontaneous foci, 不利于统计转染的
foci 数量。
谢谢！ |
|
b********g
发帖数: 46 | 18 请教，
最近遇到个棘手的问题，如何把plasmid transfer to neural stem cell? 查到一些方
法，如1)直接incubation plasmid with cells 2)电击 3）virus。不知道如何使用
virus transfect these neural stem cells? 有没有方法介绍或者试剂盒？ |
|
r******k
发帖数: 446 | 19 我最近也要做。 先virus package 然后再transfect。 如果你有兴趣咱们可以讨论一
下。 我找个protocol给你也行。 |
|
r******k
发帖数: 446 | 20 如果按着lipo2000说明书上写的 在90%左右 cell confluence的时候做transfection，
那第二天细胞就长的满满的了，这能行吗？ 是不是有contact inhibition？等细胞长
的很满了， 我可否trypsinize 然后转到更大的dish里面养？ |
|
j****t
发帖数: 1663 | 21 恩，293是长得比较快。下次铺细胞是计一下数，确定你铺的是70-80%confluence
另外，还有你也可以试试其他的transfection reagents 来提高转染效率，例如CaCl2
， PEI (Polyethylenimine），Turbofect from Thermo-Fermentas |
|
r******k
发帖数: 446 | 22 小弟最近想看一个蛋白的half life， 但是是一个mutant的蛋白。所以一般细胞系里面
没有，所以只能overexpress这个蛋白到cell line里面。 请问大家一般都transfect
24hr 还是48hr后用CHX treat 看这个蛋白的half life呢？多谢 |
|
r******k
发帖数: 446 | 23 用U87做transfection， 感觉第二天漂了不少细胞呀。 比293t 耐受能力差那么多、/
？ 大神们有啥经验不？ |
|
r******k
发帖数: 446 | 24 好 谢谢！ 我这回把细胞铺的condense不少， 也在transfection 6hr后换了液体。 基
本没怎么死。 |
|
c*******s
发帖数: 29 | 25 Xfect from Clontech is relatively the best in our hand.
transfection |
|
|
B***v
发帖数: 113 | 27 我打算做个DNA transfection，不同于传统的plasmid，我打算用最小的DNA片段
promoter--kozak--ORF (ATG to TGA)
这个是不是就可以了？有神么问题没？蛋白表达会不会受影响？ |
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p*****r
发帖数: 361 | 28 请问chemist去做liposome transfection的职业前景如何？ |
|
c********n
发帖数: 225 | 29 实验流程第二步：
2. Transfection
更新前
2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV
Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl
in 0.5x TE buffer (5 mM Tris-HCl, 0.5 mM EDTA，pH 8.0).
2-2 5’-phosphorylated ssDNA guides are dissolved to 100 ng/μl in 0.5x TE
buffer (PH 8.0). For each well of a 24-well plate, 200-250 ng NLS-NgAgo
plasmid and 100-300 ng guidesare diluted in 50 μl Opti-MEM (Gibco); 1.25 μ
l lipofectamine 2000 is diluted in 50 μl Opti-MEM.... 阅读全帖 |
|
发帖数: 1 | 30 For those who can not access the link
I copied the content here:
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
NgAgo
15 posts by 9 authors
Davide Seruggia
Jun 23
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
k*****[email protected]
Jun 24
Hi, Davide,
I tried th... 阅读全帖 |
|
发帖数: 1 | 31 Hi all,
Does anyone have some exciting result using NgAgo. I have transfected the
293 cells with NgAgo and phosphorylated ssDNA targeted two genes, however,
there is no Indels at all. Even I did multiple transfections of ssDNA, no
positive result.
- 隐藏引用文字 -
On Tuesday, June 21, 2016 at 1:39:46 PM UTC-7, Aidan wrote:
Hi all,
I am trying out the NgAgo system in hard to transfect cells in which the
CRISPR-Cas9 system never produced any edits. I'm hoping for a non-coding
large deletion mediated by ... 阅读全帖 |
|
m*********D
发帖数: 1727 | 32 （CRISPR的背景资料请参阅Feng Zhang的“Genome engineering using the CRISPR-
Cas9 system”，Nature Protocols 8, 2281–2308 (2013)）
背景
我们是作一个与激素相关的肿瘤的。这个肿瘤的治疗方法已经相当完善，就是让身体里
不能生产激素或用一个小分子化合物抢占激素受体上激素的结合部位。但多年治疗后，
会有相当比例的病人产生抗药性。这个抗药性机理相当复杂。我们实验室一直致力于筛
选和现有药物不同的机理的小分子化合物，包括受体和DNA的结合或其他与受体相关的
pathway的inhibitors。前几年成功地筛选出了几个不同机理的化合物，个别在细胞水
平达到了nM的级别，动物测试也在10 mg/kg的水平。
最近在这个领域对抗药性肿瘤的研究发现，部分病人的抗药性是因为受体激素结合部位
发生了突变。一个或几个氨基酸的突变就能导致肿瘤细胞的生长不能被现有药物抑制。
所以，我们很想知道我们筛选出来的化合物对这些突变受体是不是也起作用。理论上来
说，我们的化合物不和激素竞争，突变不会影响我们化合物的效果。但只... 阅读全帖 |
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p****y
发帖数: 95 | 33 PEI Transfection
Transfection:
1. Split 293T cells one day before transfection in DMEM/10% FBS medium:
a. 6 well dish: 0.5x106 cells
b. 10cm dish: 4.0x106 cells
c. 15cm dish: 9.0x106 cells
2. Prior to transfection bring all reagents to room temperature.
3. In a sterile tube dilute total plasmid DNA (ug) in serum-free DMEM w/o
phenol red (volume of media is 10% of final volume in culture vessel). Use
transgene: viral packaging (psPAX2):viral envelope (pMD2G) constructs at 4:2
a.... 阅读全帖 |
|
n**********g
发帖数: 196 | 34 1) Do you understand statistics?
2) you need to address why they conflict with each other. (Fig 4a and 4e).
You need to read others’ question first.
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
3) you need ... 阅读全帖 |
|
s******y
发帖数: 28562 | 35 The critical things is the plasmid without a SV40 replica site will
not be replicated in 293T cell and therefore will be "diluted"
after each cell passage.
Usually the thumb of rules are keeping the transfected cell of at
least 10% of the total cells.
293T cells are usually very easy to transfect so we can assume 90%
are transfected in the begining. However if the transfection of your protein make the cells grow slower, the untransfected cell will out-grow the transfected ones in a few days. As |
|
s******y
发帖数: 28562 | 36 For the first question, transient transfection and expression requires cell
cycle and cell replication.
The KD of your protein A or, the toxic effect of the siRNA, may affect the
cell status, therefore lead to irregular transfection efficiency.
Luciferase reporter is less likely to affect transfection efficiency.
Therefore your second method is probably better. However this is a potential
problem with the second method: are you sure you can KD protein A by such a
short treatment? Also, your luci... 阅读全帖 |
|
发帖数: 1 | 37
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
This is a whole academic issue and how it is running right now. If the rule
changes, I guess the quality of scientific research will be improved.
However, who is go... 阅读全帖 |
|
c********n
发帖数: 225 | 38 实验流程第一步：
1. Cell culture
更新前
293T cells are maintained in high-glucose DMEM (HyClone) supplemented with
10% FBS (HyClone) and penicillin/streptomycin at 37°C with 5% CO2
incubation. Cells are seeded to 24-well plate (Costar) with 60% confluence 8
hours before transfection. Thirty minutes before
transfection, cells are washed twice with PBS and medium is changed to high-
glucose DMEM medium containing 2% FBS.
更新后
293T cells are maintained in high-glucose DMEM (Gibco) supplemented with 10%
FBS (Gi... 阅读全帖 |
|
R*s
发帖数: 2041 | 39 for stable transfection: u include a selective marker (such as G418,
puromycin) in the DNA construct will transfect cells with. Then by
including the antibiotics (G18, puro...) in the media, you will be
able to seletively grow up the cells trasfected with your construct, while
killing the untrasfected ones.
For transient transfection: you don't have such selective markers in
the constracuts. You will always grow cells in regular media. Since it
is impossible to achieve 100% transfection, there i |
|
z*******a
发帖数: 175 | 40 I used solid tissue of animals. You are talking about binding of
exogenously transfected protein to
endogenous regulatory elements. I am wondering how you do transfection
efficiency control in the first place
if it is transient transfection. equal exogenously expressed protein makes
your data comparable between
treatments and repeats. I do NOT have confidence to get evenly transfected
efficiency all the time.
greater
only |
|
q**********0
发帖数: 335 | 41 Recently, I transfected pmaxGFP plasmid to HepG2 cells by using
Lipofectamine2000 to track the positive-transfected cells with another test
plasmid. However, the FACS result showed no separation between GFP positive
or negative cells, only one cell population. I don't know whether the
transfection efficiency is too high that almost every cell was transfected?
or pmaxGFP is not a proper GFP plasmid to use in FACS? Your suggestions will
be appreciated. |
|
i*****i
发帖数: 154 | 42 手头所有的cell line都非常难转，用LP2000也就10-20%的transfection。
但是又必须提高transfection效率，所以想买一个电转仪。
自己对这方面没有经验，希望版上牛人推荐一个比较好用的，尤其是对prostate cell
line效率高的电转系统。
不知道大家对下面的系统评价怎么样？
Life technologies的
Neon® Transfection System for Electroporation
BTX的
ECM 830 for Adherent Cell Transfection |
|
发帖数: 1 | 43 I was a nucleic acid research scientist, but not working on gene editing any
more
. The reason I am involved is to present some basic knowledge on nucleic
acid
research.
{1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
it.
The biggest concern is not addressed by your answer. }
Band shift sometimes can be seen due to sample ionic strength and buffer.
{ 2) DNA band and migration are easily alternated by sample ionic strength
and
buffer.
No, I did not talk about band dist... 阅读全帖 |
|
n**********g
发帖数: 196 | 44 1) The current low (zero) verified success rate is sufficient for people to
question the story.
When will your statistic data collection be completed? Let's talk about the
success
rate then.
Your comments are so interesting. If you do not know the science, just be
patient and wait for more answers.
2) “Do you think 1.3 mm difference (resolution)
can be achieved on peaks with a width of 1.5-2 mm?” The answer is yes
because of Fig 4e. Your statement is “difference
sample preparation” is completely... 阅读全帖 |
|
m****s
发帖数: 18160 | 45 2016年5月2日，英国《自然》杂志子刊《自然 生物技术》刊载了一篇有关"基因编辑工具"的论文，论文负责人的英文署名是"Chunyu Han"，这篇论文在华人生物界获得广泛赞誉。论文发表的第二天，韩春雨的名字和论文在一个名为"生物艺术全球生物医学交流"的微信群里得到了热烈讨论。生命体从受精卵开始，经过不断分裂，可分化出大约60兆亿个构建人类身体的细胞，每个细胞都含有全部生命遗传信息的基因组，所谓基因组编辑技术，就是人为来调整、改变、插入、去除、修改个体或物种的基因组序列。
http://tv.cctv.com/2017/05/20/VIDEBZRf7rQNg2Y1ExpBhpQM170520.shtml
一、学界和媒体的热议Mitbbs.com
一年前，英国《自然生物技术》杂志刊载了一篇有关"基因编辑工具"的论文。论文的负责人"一鸣惊人"，得到了学界和媒体给予的无限荣光。在《自然》杂志社的所属期刊上发表论文，通常表明，该科研成果走在了世界科学研究的最前沿，并有可能对未来科学的发展起到关键性作用。此消息一经媒体爆出，在站内引起站内相关专业人士的热议。
国内科研水平不得了了，河北科技大学在... 阅读全帖 |
|
m****s
发帖数: 18160 | 46 2016年5月2日，英国《自然》杂志子刊《自然 生物技术》刊载了一篇有关"基因编辑工具"的论文，论文负责人的英文署名是"Chunyu Han"，这篇论文在华人生物界获得广泛赞誉。论文发表的第二天，韩春雨的名字和论文在一个名为"生物艺术全球生物医学交流"的微信群里得到了热烈讨论。生命体从受精卵开始，经过不断分裂，可分化出大约60兆亿个构建人类身体的细胞，每个细胞都含有全部生命遗传信息的基因组，所谓基因组编辑技术，就是人为来调整、改变、插入、去除、修改个体或物种的基因组序列。
http://tv.cctv.com/2017/05/20/VIDEBZRf7rQNg2Y1ExpBhpQM170520.shtml
一、学界和媒体的热议Mitbbs.com
一年前，英国《自然生物技术》杂志刊载了一篇有关"基因编辑工具"的论文。论文的负责人"一鸣惊人"，得到了学界和媒体给予的无限荣光。在《自然》杂志社的所属期刊上发表论文，通常表明，该科研成果走在了世界科学研究的最前沿，并有可能对未来科学的发展起到关键性作用。此消息一经媒体爆出，在站内引起站内相关专业人士的热议。
国内科研水平不得了了，河北科技大学在... 阅读全帖 |
|
J*****w
发帖数: 180 | 47 我朋友正在帮国内厂家物色有领导能力的，有创业精神的专家，在国内药厂组建蛋白质
药物开发部。
A leading China Pharma Company has immediate openings for the following four
leadership positions in its Yantai, China and Singapore sites. Please send
your resume to [email protected] if interested.
Title: Head, Upstream Process Development
Functional Description:
Lead, build and develop a high performance Upstream Process Development
group engaged in mammalian cell culture development for commercial
therapeutic proteins, including subclone/clone sel... 阅读全帖 |
|
c******n
发帖数: 16666 | 48 【 以下文字转载自 Biology 讨论区 】
发信人: mitbbs (未名空间), 信区: Biology
标 题: Mitbbs水平挺高的，纯从科学家角度质疑韩春雨
发信站: BBS 未名空间站 (Sun May 21 23:40:17 2017, 美东)
2016年5月2日，英国《自然》杂志子刊《自然 生物技术》刊载了一篇有关"基因编辑工具"的论文，论文负责人的英文署名是"Chunyu Han"，这篇论文在华人生物界获得广泛赞誉。论文发表的第二天，韩春雨的名字和论文在一个名为"生物艺术全球生物医学交流"的微信群里得到了热烈讨论。生命体从受精卵开始，经过不断分裂，可分化出大约60兆亿个构建人类身体的细胞，每个细胞都含有全部生命遗传信息的基因组，所谓基因组编辑技术，就是人为来调整、改变、插入、去除、修改个体或物种的基因组序列。
http://tv.cctv.com/2017/05/20/VIDEBZRf7rQNg2Y1ExpBhpQM170520.shtml
一、学界和媒体的热议Mitbbs.com
一年前，英国《自然生物技术》杂志刊载了一篇有关"基因编辑工具"的... 阅读全帖 |
|
O******e
发帖数: 4845 | 49 ☆─────────────────────────────────────☆
icepiao (icepiao) 于 (Sun Sep 17 00:44:40 2006) 提到:
我转染后利用Anti-V5-FITC进行荧光染色，买的是invitrogen的抗体，目的是看
plasmid transfection后一个protein的表达情况，V5是该蛋白的Fusion protein tag
，因此只要该protein表达，理论上V5应该也表达，基因的启动子是CMV启动子。
步骤为首先co-transfection，利用另外一个report plasmid看transfection
efficiency, 48小时后PBS洗两遍，利用100%甲醇固定5min,然后PBS洗5遍，2min每次，
加入含10%胎牛血清的PBS Block20分钟，然后加入荧光抗体，置暗处1h,然后荧光显微
镜下观察，但是在荧光显微镜下几乎什么也看不到，偶尔有几个有可能有荧光的细胞，
但是太少了。
随后我也采用了4% PFA fix 0.1%Triton x-100 permeabilize, |
|
j********r
发帖数: 156 | 50 Admiring yi xia! It seems that your efficiency is much higher than the two
NS papers using non-viral vector. Just curious which MEF did you use (to
enable transfection and drug selection)? I also tried plasmid transfection (
via lipids) in primary MEF (at passage 2), however, the transfection is
extremely low. Thanks for the great information! |
|
