a****d
发帖数: 1919 | 1 for Ca2+ P method, make sure the pH~ 7.04; after adding the mixed plamids,
buffer, wait only half hour, then wash out the transfection medium as clean
as possible(you should be able to see only the black aggregates on the cell
surface, but not other places), then add fresh warm culture medium.
You can also try Fugene HD from Roche, which is better and less toxic
compared to lipofectamin and similar products.
you can also grow cortical neurons on top of glia cells to preserve activity
and help ce |
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p*****y
发帖数: 44 | 2 Transfect HEK cell to produce retro virus. 260:280=1.9 Qiagen大提的质粒
效果不怎么好，尤其试用VSVG做envelope protein, 请问有没有别的方法提高DNA纯度？
文献说CSCL最好，但是太麻烦。试过醋酸钠重新沉淀，酚，氯仿抽提一下，纯度没见到
变化，再转染，不产生病毒了！
求其他纯化方法！ |
|
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s******y
发帖数: 28562 | 4 大概可以弄到20%的样子。
如果你要更高的话就得用电转了 |
|
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v*******a
发帖数: 759 | 6 how about lenti viral shRNA vector? |
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a****l
发帖数: 125 | 7 我要knockdown 的蛋白对cell proliferation 很重要。害怕用lenti-shRNA 后细胞不
长了。所以想用transient 的siRNA. 但具体Lenti-shRNA 会不会造成这种后果也不清
楚。还请在坐赐教。 |
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g******r
发帖数: 139 | 8 我曾经试图在MEF里knockdown一个人细胞里很容易作到的基因，结果不管用电转还是
Invitrogen的2000或者RNAiMAX都不行。后来我投稿的杂志编辑也承认这是很难的 （
knockdown in MEFs is often challenging）。 |
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j*****q
发帖数: 82 | 9 lz如果你能保证second infection插入的copy是在第一次infection的同一个位置，还
是有机会提高protein level的。double transfection有人用来提高protein产量的。 |
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m*********n
发帖数: 215 | 10 谢谢楼上各位分享经验！
我们实验室刚刚开始做ChIP-seq，有一个学生做这个，说是揣摩了很久的protocol。
我plate了25million N2A cells给他，他还说Chromatin浓度不够。我吐血。。。因为
transfection看起来很好，细胞很健康。就算撑死细胞死了5million吧，那还有
20million活着呢。因为我自己不做这个，所以也不是很懂。但是我总觉得>20 million
cells怎么可能不够。如果做ChIP的话是more than enough。虽然说ChIP-seq大概需要
更多的chromatin，但是因为表达的蛋白是HA-Tag的，HA的抗体非常非常好，所以
Chromatin的浓度要求应该比一般蛋白的要低。
我就是很不明白为什么总是说Chromatin不够。前面说用Primary culture做,我Culture
了4个litter（35个胚胎）的小鼠皮层，那个学生也说是细胞不够。。。所以我觉得不
对劲。但是老板也说不出个所以然来。实验拖拉着我急死了。
不知道前辈用的是什么Protocal呢？可以分享下吗？ |
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h****n
发帖数: 2552 | 11 Transient transfection后你的蛋白主要在胞质还是在核里面？有些蛋白over express
后会change localization |
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j********r
发帖数: 156 | 12 Please recommend a lipid for primary MEF transfection. Thanks,
Seems electroporation (amexa) gives the rate around 15%, who might be quite
low. There are many lipo products on the market, just name a few
lipofetamine 2000 (with plus)
Fugene 6
GenePorter
GeneCarrier
DreamFect
Superfect
....
which or what else is best for MEF? |
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s******y
发帖数: 28562 | 13 How come? Such thing can be done in vitro in a rather straight forward way.
Can we transfect the donor bone marrow cells and then go for immuno
stimulate
and selection and finally select for the memory B cell that express that
particular antibody and put it back to the donor's body?
I mean there will be a lot of work invovled but in theory that should be
doable
right? |
|
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o**4
发帖数: 35028 | 15 Oh,I see
It's very good to know this.
Thanks again
protein make the cells grow slower, the untransfected cell will out-grow
the transfected ones in a few days. Assuming the 293T cells replicate every
24 hours, then in three days, the 10% untransfected cells will become 8
times of their original number and therefore will reach almost 80% of the
total cell population. |
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R*****w
发帖数: 447 | 16 学习了，
请问怎么知道plasmid 是否有SV40 replica site? pcDNA3.1 有SV40 replica site吗？
protein make the cells grow slower, the untransfected cell will out-grow
the transfected ones in a few days. Assuming the 293T cells replicate every
24 hours, then in three days, the 10% untransfected cells will become 8
times of their original number and therefore will reach almost 80% of the
total cell population. |
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s******y
发帖数: 28562 | 17 Did you transfect your cell with a plasmid?
In such a case, you need to treat the RNA sample carefully with DNaseI to
remove all the plasmid DNAs. Otherwise you are just doing PCR using the
plasmid as template |
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x******i
发帖数: 918 | 18 CHO-S (s means suspension) is popular in industry and very easy for
transfection.
you can purchase from Invitrogen |
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C****7
发帖数: 270 | 19 我想卖的物品:
Bovine Pit. Extract (BPE)
Anastrozole
Charcoal/Dextran Treated Fetal Bovine Serum
Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (4.5 g⁄L D-
glucose)
RPMI Medium 1640 (1X), liquid.Contains L-glutamine, but no phenol red.
GAPDH siRNA
X-tremeGENE siRNA Transfection Reagent
Opti-MEM® I Reduced-Serum Medium
可接受价格(必须明码标价！):
Up to 60% off original price
物品新旧要求:
邮寄方式要求:
You chose you pay
买卖双方谁承担邮寄损失(Required if not code only):
Before mail on me
After mail on you
付款方式说明: |
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s*******g
发帖数: 121 | 20 try transfection kit from Amaxa or shRNA in lentivirus.
20nM |
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w***a
发帖数: 4361 | 21 跟siRNA本身可能根本没关系，就是transfection效率低下的问题，
用cocktail的话引入更多off target。
这种primary cell本来就很难转化，你试试电转看怎么样。
20nM |
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W****C
发帖数: 1937 | 22 基本所有的macrophage transfection效率都很低 |
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c**n
发帖数: 73 | 23 try CaPO4 transfection.
experiment? |
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v***a
发帖数: 1242 | 24 Yes, I did. But my boss required that the result from empty vector control
be the same as the normal control to keep the consistence... How can I
explain it solidly enough to convince my boss? Or in fact I did something
wrong that the influence of the empty vector control should not be that much
(made the mRNA level of my protein nearly undetectable)? |
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v***a
发帖数: 1242 | 25 谢谢！！！我承认你说得都对，I've got a long, long way to go
of
point
u |
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h****g
发帖数: 439 | 26 是不是这两种质粒的抗性不能相同。 其中一个质粒整合了，不意味着另一个也整合吧。
还有没有其他的特殊要求？哪位大虾有经验？谢谢 |
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g*****y
发帖数: 6325 | 27 2个vector抗性必须不同，先转一个再转另一个。 |
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h****g
发帖数: 439 | 28 您的意思是要先转一个，筛出stable的，然后再转另外一个吗？
不能同时转，一块筛吗？ |
|
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s******y
发帖数: 28562 | 30 可以同时转，但是这样效率很低，很难选出双阳性。除非是用病毒转。 |
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s******y
发帖数: 28562 | 31 另外，抗性是否相同无所谓，只要你能找到其他筛选方法（比方说用荧光）
吧。 |
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a***e
发帖数: 1010 | 32 why not clone your target gene into one vector? |
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h****g
发帖数: 439 | 33 Good point. because I already got these two vectors. |
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h****g
发帖数: 439 | 34 谢谢gysonny和sunnday ，决定先转一个，再转另一个了 |
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a***e
发帖数: 1010 | 35 Both siRNA and lentiviral shRNA methods are good enough to reach your
goal.
However, lentivirus usually has a higher infection efficacy than
transfection method. Therefore, using lentiviral shRNA may be a more
efficient
way to knockdown gene expression. |
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a***e
发帖数: 1010 | 36 I agree that it may be not that important to treat RNA sample with DNaseI
during the RNA purification process in the original question. However, it is
better to maintain a "good habit" to do so.
There are several reasons for this:
(1) it doesn't add many extra work. DNaseI treatment is very simple and fast
. Qiagen has on-column DNaseI digestion kit. Or an extra TRIzol purification
step only adds one more hour.
(2) if the experiments include transfection of plasmid to overexpress some
genes or s |
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m***n
发帖数: 337 | 37 我在mapping一个protein (A)和另外一个protein (B)相互作用的区域。首先wt A 和B
是有相互
作用的。 我发A的predict的6个domain分别表达，然后和B co－transfection， IP显
示单独的
6个domain每个都可以和B相互作用。 我的negative和positive IP control都没问题。
结果让
我很难相信，我也不知道是什么原因。大家有没有遇到类似的问题，any suggestions
and
explanations would be appreciated. thank you so much |
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a***e
发帖数: 1010 | 38 Since you are doing co-transfection to overexpress two proteins
simultaneously, it is quite possible to bring in such false positive outcome. |
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s******y
发帖数: 28562 | 39 我不知道这个蛋白是否会泛素化。
但是，蛋白泛素化一般是不能直接看到smear的，因为泛素化的比例一般都不超过该蛋
白的1%。要看smear一般需要同时transfect 6His-Ub, 或者HA-Ubiquitin,
然后要么pulldown his tag and then use your protein antibody to check the
smear, or pulldown your protein and then use HA antibody to check the smear |
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z****g
发帖数: 3340 | 40 If you don't have special preference to make a stable in HeLa cells, just
switch to 293 cells which is much
much easy for transfection, selection and colony picking.
also you can make some extra plates and check expression of your gene after
selection at different time
points. so you may get idea how is your transgene expression along with long
-term selection.
in
line? |
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a*********7
发帖数: 342 | 41 I think it is really important to do a RT-PCR to know if the target gene is
expressed or not. During stable transfection it is quite often that part of
you target gene will not get integrated into cellular genome because this
is a purely random integration process. If you can detect the RNA
expression, you might be more confident about protein detection. If
necessary, you can even enrich the protein by IP before western blot. |
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M******t
发帖数: 555 | 42 试过lipofectamine／2000，效率太低了。谢谢。 |
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j****g
发帖数: 406 | 43 我的经验是不同的cell line有不同的最合适的reagent。
293T, Hela用lipofectamine／2000效果很不错
NIH/3T3用RNAiMAX from invitrogen following the reverse protocol
N2a听说roche的X-tremeGENE不错
另外DHARMACON的1，2，3，4也有很多不同的最适合的cell line. |
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v***a
发帖数: 1242 | 44 我用的是NOVAGEN的RIBOJUICE，还不错 |
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z****i
发帖数: 35 | 47 Macrophage, DC, 我们用的是polyplus的INTERFERin,效率可达80% |
|
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S*****s
发帖数: 287 | 49 谢谢楼上各位。我正在看这方面的文章，特别是七楼 jcb 老兄列出来的那篇，越看越
觉得不错。我做的蛋白目前没有 endogenous cell line，而我研究的方向也包括 mRNA
intron splicing。现在我都是用引入了 CMV promoter 的 BAC DNA 做 transfection
。看看这些文章我倒是想买点这个 ZFN 然后把 HEK293T 细胞的基因组改一改，改造出
一个可以 endogenous cell line 来。明天和 sigma 的 rep 继续谈谈。 |
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m****n
发帖数: 1066 | 50 RNAi,in vitro transfection.
Control and siRNA treated, QPCR data.
Which one to use, equal variance or unequal variance?
Thanks. |
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