o**4
发帖数: 35028 | 1 我转染的时候细胞密度有点低，
转染之后细胞又生长很慢。
现在已经快72个小时了，
还能让细胞再生长么？ |
s******y
发帖数: 28562 | 2 If your plasmid has SV40 replicate sites and if the expression doesn't kill
the 293T cells, then I think you can grow them for a week or more.
【在 o**4 的大作中提到】
: 我转染的时候细胞密度有点低，
: 转染之后细胞又生长很慢。
: 现在已经快72个小时了，
: 还能让细胞再生长么？
|
o**4
发帖数: 35028 | 3 No SV40 replicate sites
kill
【在 s******y 的大作中提到】
: If your plasmid has SV40 replicate sites and if the expression doesn't kill
: the 293T cells, then I think you can grow them for a week or more.
|
s******y
发帖数: 28562 | 4 Then you can only keep them for up to the double number of cells
In most case it only take one day for the 293T cell to grow to double number.
In your particular case you will have to judge by yourself
【在 o**4 的大作中提到】
: No SV40 replicate sites
:
: kill
|
o**4
发帖数: 35028 | 5 It works for me in 2 or 3 days.
I just don't know whether it works more than 3 days.
Anyway, thanks a lot.
number.
【在 s******y 的大作中提到】
: Then you can only keep them for up to the double number of cells
: In most case it only take one day for the 293T cell to grow to double number.
: In your particular case you will have to judge by yourself
|
s******y
发帖数: 28562 | 6 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
【在 o**4 的大作中提到】
: It works for me in 2 or 3 days.
: I just don't know whether it works more than 3 days.
: Anyway, thanks a lot.
:
: number.
|
o**4
发帖数: 35028 | 7 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.
【在 s******y 的大作中提到】
: 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
|
n***w
发帖数: 2405 | |
v***a
发帖数: 1242 | 9 同仰慕
【在 n***w 的大作中提到】
: 学习了。
: 觉得sunny前辈好厉害。。。^^
|
R*****w
发帖数: 447 | 10 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.
【在 s******y 的大作中提到】
: 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 | 11 看一下vector map或者说明书，上面有的。
要知道这个sv40怎么来的不妨自己google 一下
并不是每一种细胞都有这个功能的，293T 细胞是因为被某种病毒感染了所以才有
这种功能
我刚才给他算的那个答案是针对他的具体情况算的。对于别的情况要修改一下
算法.
不过一般来讲是不应该超过3天。
吗？
every
【在 R*****w 的大作中提到】
: 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.
|
R*****w
发帖数: 447 | 12 Thank you very much, sunnyday!
I figured it out and found that pcDNA3.1 does have sv40 origin.
The following is some information from Wiki, in case someone else is
interested in this:
HEK 293 cells were generated in early 70s by transformation of cultures of
normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex
Van der Eb's laboratory in Leiden, Holland.
An important variant of HEK293 cell line is the 293T cell line that contains
, in addition, the SV40 Large T-antigen, that |
