v***a
发帖数: 1242 | 1 transfection的efficiency是否跟cell density相关呢？细胞少了是不是efficiency就
高，但是toxicity会大？
我做plasmid DNA transfection的时候，发现普通control（没有加任何transfection
reagent的）和有vector plasmid transfection的control相比，我要观测的蛋白表达
不一样，有vector的蛋白少了很多。不知道这是怎么回事呢？
谢谢～ |
|
n****y
发帖数: 819 | 2 请问 siRNA transfection, 我用的是dharmacon的transfection reagent, 6 well
plate, 每一孔我用了4 ul, 好像再多的话细胞很快就死了， 然后siRNA 从10 nM 到
100 nM 都式过。 我的问题是， transfection reagent 和 siRNA之间是不是应该有一
个固定的比例呢？ 比如 10 nM siRNA用 4 uL transfection reagent, 20 nM 用 8 uL
? 多谢！ |
|
m*p
发帖数: 226 | 3 Embryonic hearts were co-transfected by RFP and GFP plasmids. Only 20% were
transfected. Boss argued that
all RFP and GFP should co-localized and I said it is impossible, only when
RFP and GFP plasmids are in the
same lipid vesicle and fused with the same cell, then they will colocalize.
She said she was one thousand
percent sure. I can not say more, as this might harm the relationship.
However, I would like to know the truth about this co-transfection. Thanks
for your imput. |
|
A****w
发帖数: 244 | 4 BD BaculoGold DNA ＋ pVL1393-DNA 那个没有bacmid prep的这一步
bac to bac BACMID DNA用M13和目标基因的特异引物检测的，可以看到pcr产物，难道
是transfection的量不够，用quick and dirty prep的。
》》是不是根本就没有拿到recombinant baculovirus啊？我们做的时候7天以后Sf9
cells
就好多死细胞了。Sf21应该也一样啊。
感觉是没有拿到baculovirus，细胞transfection 5－7天后，看不到涨大的病态细胞（
或者和control差别不大）看不出来。。。
有些时候看到是好多死细胞了，有的时候看起来还行。
》》“amplification后也没有”是什么意思？没有检测到目标蛋白基因的表达？
就是在transfection后，有这个supernatant去infect健康的细胞（在T75里）。然后看
细胞形态，也做蛋白表达检测（western） |
|
r**********e
发帖数: 587 | 5 I don't know anyone here has experience with plasmid transfection fir SH-
SY5Y?
Lipofectamine 3000 doesn't work at all.
Neuronal line is always challenging for transfection.
Any recommendations of good reagent to achieve relatively high transfection
efficiency?
Thx! |
|
C*****h
发帖数: 926 | 6 【 以下文字转载自 Biology 讨论区 】
发信人: Carwash (for+free), 信区: Biology
标 题: 3T3 transfection效率太低
发信站: BBS 未名空间站 (Wed Apr 28 21:47:39 2010, 美东)
用lipofectamine 2000转染3T3细胞，但是transfection效率太低（不到1％），
不知道是怎么回事？
0.8 ug plasmid DNA + 2 ul lipofectamine
转染1x10^5细胞。
不知道大家是怎么检测shRNA的knockdown的效果？
我是把带有shRNA的plasmid，转染到3T3细胞，然后用western blot检测蛋白质，
看目标蛋白是不是减少了。
可是现在转染效率不到1％，就算shRNA把那1％的细胞内的目标蛋白全部knockdown，
总个western blot还剩下99％的蛋白质。
有什么办法提高3T3细胞的转染率？有人转染过这个细胞系吗？请帮忙指教一下。谢谢。 |
|
R*s
发帖数: 2041 | 7 yes, immortalized cell lines are generally harder to transfect
than primary cells.
however, 283 in general should be easy to transfect unless your
genes are really toxic to the cells (some of genes I have to use
are in such case).
Some guy upstair mentioned Superfect Reagent from Qiagen.
That one is quite effective, and better than LipofectAmine
in many cases, but more expensive too. |
|
c*****t
发帖数: 1084 | 8 check the protocol of agent. some agents require highly confluent
cells to perform transfection. some require 50%. generally
overgrown cells are not good for transfection. |
|
t******y
发帖数: 716 | 9 if you mixed well, you should be able to see what your boss expected. The
reason is simple that both plasmids have the same chance binding to every
single lipofectamine 2000 particle(if you're using it as transfection
reagent).Each lipofectamine 2000 particle probably can bind a bunch of DNA
and sometimes the particle size is pretty big,ranged from ~40 nm to ~180 nm
in diameter. There is a paper talking about DNA and transfection particles.
http://www.biomedcentral.com/1472-6750/8/23 |
|
d****d
发帖数: 214 | 10 In my hands and using TransIT-LT1 from Mirus, the tranfection efficiency of
3T3 cells can be as high as 65% for our GFP expressing reporter plasmid. The
transfection efficiency of 3T3 cells can be as high as 90% using Neon
electroporation system, although only ~20% cells survived the procedure. The
transfection efficiency is derived from flow cytometry data. It could be
lower if you just look at the cells under microscope. |
|
a****l
发帖数: 125 | 11 诸位大侠可知到用什麽方发transfect siRNA 到 MEFs cells 里面吗? 要 transient
的transfection 就可以. 貌似MEFs 不太好转染的说. Lipofectamin 2000 可以吗?
谢先 |
|
h***a
发帖数: 145 | 12 Hi,
I am wondering if anyone worked on MLE-15 cells transfection with siRNA
before?
What lipid solution did you use and at what confluency point did you
transfect the cells?
Thanks a lot |
|
n********k
发帖数: 2818 | 13 it is not a proper comparison, many transfection reagents impact on cells...
think about it, they are peneratring
cell membranes...
transfection |
|
v***a
发帖数: 1242 | 14 嗯，加了transfection reagent的control某些指标明显跟未加reagent的control不一
样了。那我在paper里可以这样说明吗？我老板说应该保持前后一致。比如我前面一个
蛋白在无transfection的cells里表达量有较多，但现在在有vector的control里，蛋白
的表达量下降了好多，用RT-PCR检测几乎就要看不到了；老板就要我重做，说这2个
control非得match。大家遇到过这样的情况吗？如果前后不match，该如何说明比较好？
.. |
|
n********k
发帖数: 2818 | 15 your boss is typically those ...I don;t know what to say...BTW, the level of
mRNA etc we normally detect is
always in a relative sense, I don't see either you or your boss has a point
here..., so just do another control to
see whether it is your vector or transfection reagent problems: add
transfections reagents without any DNA or
different vectors...BTW, No offense, I suppose you are a newbie, otherwise u
got quite some work to do with
your design and interpretation of experiments and troublin |
|
g*****y
发帖数: 6325 | 16 cell density在70%-80%的transfection的efficiency 最高
transfection |
|
f**********s
发帖数: 1415 | 17 I plan to transfect GFP-fused target protein to my cell line and establish
stable cell line. I am wondering if there is neccessary to include negative
control (like salmon sperm DNA) or positive control (vector without encoding
GFP or target protein) during the transfection and selection process.
Thanks!
|
|
s*****g
发帖数: 7857 | 18 我按照Lonza/Amaxa的program做transfection of suspension cells.当时过夜后的细
胞存活率是60%.可是加抗生素选择后几乎就死光光了.即使几天后有20%的细胞,也最后
走向死亡.
有做着方面的专家,给分析一下,是为什么?
以前都做附着细胞的transfection.过夜后活细胞都附着培养皿底部.把含死细胞的培养
液换成新培养液.再加抗生素/培养液就可以得到想要的但细胞克隆了. |
|
f**********s
发帖数: 1415 | 19 for plasmid transfection to establish stable transfection. |
|
z*******6
发帖数: 679 | 20 I don't think the selection marker matters a lot. I think it is the cell
line and the way of transfection that matters more. If you don't care the
expression level tooooo much, lentivirus works really good. But last time I
was doing A549 and I need the expression level to be really high to secrete
a lot of soluble proteins for ELISA detection, none of methods (
electroporation, nucleous transfection, lentivirus) or selection markers (I
tried multiple ones) worked. |
|
n******u
发帖数: 418 | 21 只用过10T1/2
看你transfection后做什么了，luc assay的话，用ca2+-mediated transfection都可以
一般的话，用fugene6好了 |
|
|
f*c
发帖数: 2726 | 23 almost all the transfection protocols (mammalian adherent cells) say: split
cells one day before transfection. Why? why should be one day ,not two days
or more?
thanks |
|
A****w
发帖数: 244 | 24 最近在做baculovirus transfection一直做不出来
用了两种不同的方法，一个是BD BaculoGold DNA ＋ pVL1393-DNA.
就是follow manual
另一是Bac to Bac system， 用cellfectin II reagent。
用的是Sf-21 conditioned in Sf-900II media （大于95％viability)
怎么做都在5天transfection后看不到病态细胞
然后amplification后也没有～
请大牛指教！
感谢！ |
|
j******i
发帖数: 939 | 25 你记错了吧 HeLa是有名的hard to transfect的细胞 大概只有10-20%能transfect到 |
|
j****t
发帖数: 1663 | 26 我一般是70-80%confluence的时候做transfection，这个也要看什么样的细胞，有些细
胞分裂速度慢，多铺一些细胞应该也没有关系。
如果你已经用lipo2000做了transfection，我觉得第二天就trypsinize不太好。
建议你下次转一个带荧光的质粒，看看转染效率，这样你就知道铺多少细胞最好了。 |
|
r******g
发帖数: 600 | 27 我做lipo transfect MCF-7
6 well plate- 2ml culture/well
一下protocol剂量 用于 one sample (1 well)
12.5 lipo diluted in 150uL OPTI-MEM
2.5ug plasmid DNA diluted in 150uL OPTI-MEM
sit in RT for 20'
把上面的两个150uL Mix 约等于300uL
sit for 5'
250uL used for each well for transfection. |
|
j*****9
发帖数: 716 | 28 I have tried about 10 lipid-based transfection reagents in SH-SY5Y and
neurons. None of them gave me a decent efficiency. NeuroPORTER (Genlantis or
Sigma-Aldrich) was the best (~30% in SH-SY5Y, 18% in Neurons using FACS).
Good luck
transfection |
|
d***e
发帖数: 243 | 29 我来补充一下,不是查google的,是三年前在北医帮导师写教材时我的一些理解.
Transfect一般是指各种artificial的手段给动物细胞内导入外源的基因片段.
Transduction ,conjugation涉及的是细菌间的基因转移,不关真核细胞的事.
1. 转化(Transformation).受体菌直接摄取供体菌游离DNA片段,转化的DNA片段可以是细
菌溶解 释放,也可以是人工提取. 摄取能力称为感受态 competence.应用吗,可以讲是最
早证实了DNA是遗传物质的Griffith的肺炎球菌实验.
2.转导 (Transduction). 温和噬菌体介导的遗传物质从供体菌到受体菌的转移.这里强调
的是temperate phage, 不是指溶原性噬菌体lysogenic phage.作用是细菌自发获得一些
新的protein,eg. toxin or antigen.
3.接合(Conjugation). 是plasmids介导的细胞间接触,是供体菌遗传物质进入受体菌.供
体菌遗传物质不一定就非是供体菌的gene,也可能只是供体菌的质粒transfer到rece |
|
c**a
发帖数: 94 | 30 以前没有用过昆虫细胞,现在需要从SF9 细胞里表达蛋白并纯化. 之前试着用
lipofectamine
tranfect细胞, 没有收集到多少蛋白. 请求有经验的高手给个protocol或者方法,用什
么transfection
reagent比较好. 怎么lyse和纯化蛋白. 谢谢了. |
|
J********n
发帖数: 534 | 31 这个不用transfection reagent。
需要先制备P3 virus (就是P1 amplify三次)，sf9 cells很容易break，比E.coli更简
单。之后最好ultracentrifuge 40K, 1hour.
要更具体的，上invitrogen或者BD Biosciences看看。他们产生P1 virus所用方法不同。
当然，比较新的BacMam就更简单了。 |
|
c**a
发帖数: 94 | 32 要用virus吗, 我们现在没有任何virus系统,只能做transfection
同。 |
|
a*****n
发帖数: 2835 | 33 transfection用293T吧
sf9是做病毒比较好 |
|
c**a
发帖数: 94 | 34 是, 但如果 不得不在 sf9 上做transfection呢, 有没有好的建议啊 |
|
C*****h
发帖数: 926 | 35 用lipofectamine 2000转染3T3细胞，但是transfection效率太低（不到1％），
不知道是怎么回事？
0.8 ug plasmid DNA + 2 ul lipofectamine
转染1x10^5细胞。
不知道大家是怎么检测shRNA的knockdown的效果？
我是把带有shRNA的plasmid，转染到3T3细胞，然后用western blot检测蛋白质，
看目标蛋白是不是减少了。
可是现在转染效率不到1％，就算shRNA把那1％的细胞内的目标蛋白全部knockdown，
总个western blot还剩下99％的蛋白质。
有什么办法提高3T3细胞的转染率？有人转染过这个细胞系吗？请帮忙指教一下。谢谢。 |
|
a****d
发帖数: 1919 | 36 I use lentivirus to make sure high efficiency of transfection. |
|
c**n
发帖数: 73 | 37 You are supposed to transfect cells with empty vector as a control
好？ |
|
m*****u
发帖数: 15526 | 38 最好先过一两天让细胞缓一缓，transfected gene充分表达后再加抗生素。筛之前先做
个抗生素浓度
titration,摸个合适浓度。 |
|
c******r
发帖数: 3778 | 39 关键是，有test transfection efficiency吗？大概多高？用的什么antibiotics做的
selection？ |
|
s*****g
发帖数: 7857 | 40 test transfection efficiency没有做.因为他们网上有我细胞系的program参数。大约
在60%
用的什么antibiotics做的selection -Hygromycin （通常你们的细胞系都要多少浓度
？）我是用MTT来测titration的。
是否是由于四细胞和活细胞在一起，死细胞对活细胞有影响，而造成的呢？ |
|
w******e
发帖数: 1187 | 41 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！ |
|
i******m
发帖数: 495 | 42 俺想买个vector做transfection的positive control, 在网上搜了搜, 好象Clontech做
的不错, 不过打电话过去, 告知对于俺们这种for-profit organization, 一年要交$
10K的license fee才可以用. mmd. 不知道同学们能不能给推荐其他比较好的GFP/RFT/
etc 的来源? //BOW |
|
H****s
发帖数: 301 | 43 It depends on your purpose and the cell line to be transfected. Generally
speaking, if you cell line is tough enough, puromycin is much easier to work
with. You just need to add a relevant amount of puromycin to the media and
it starts killing.
For neomycin and hygromycin, you have to determine optimal killing
concentration specific to the cell line you will use. To initiate killing,
you have to split cells and supplement with neo/hygro containing media.
Every selective marker has its specific p... 阅读全帖 |
|
b******e
发帖数: 3348 | 44 问题可能有些弱智，老板要求转染某个基因的siRNA到一个动物细胞，以前我没做过
siRNA transfection,只用lipofectamine 2000转染过某基因的overexpress vector到
动物细胞，网上查了一下，dharmacon有这个基因的siRNA,是不是买了那个smartpool,
然后用lipofectamine 2000就可以转染了（当然成功与否，到不到50%得实验做了才知
道）,那个smartpool是四个RNAs的pool对吧，不知道这个过程是不是正确的。
菜鸟一个，敬请指教，非常感谢。如果有protocl能够让小弟看看那就更好了
包子答谢。
PS，不需要长期stock,所以暂时不考虑shRNA. |
|
c********l
发帖数: 244 | 45 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. |
|
h*****G
发帖数: 113 | 46 小弟刚开始做stem cell，现在需要用siRNA 转进mouse stem cell 去knockdown基因。
试了roche的x-treme siRNA transfection kit， 效果不理想，几乎没有knock down
target 基因。
希望有经验的大侠推荐合适的试剂，使用的siRNA的量和注意事项。
thanks～～ |
|
B******o
发帖数: 496 | 47 具体是啥 stem cell？ 查查文献别人用啥试剂做的transfection撒
down |
|
b******e
发帖数: 3348 | 48 是不是2-3天，细胞就慢慢把transfected进去的vector给排出了？还是能维持一个礼拜
左右？ |
|
f**g
发帖数: 28 | 49 Hi, everyone, is there any connection between cell cycle and transfect
efficiency. Why there are different expression level of tranfected genes in
different cells? Thanks for any suggestion. |
|
v*********d
发帖数: 382 | 50 invitrogen Neon Transfection Syste
C2C12 90%+ efficiency. Should also work for 10T1/2
Best part, you can get a 2week free demo |
|
