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Jurkat T cell转染求助# Biology - 生物学
e*2
1
I never tried to predict a VB but, as a freshman here, I did do some
homework to try to understand VB and its trend.
With that being said, the next VB is doomed meaningful, especially for those
PDs in the second half of 2008 (calendar year) and later, as this VB, after
6.5 months' leap forward, should give us some decision power to keep
looking forward or simply turn away from this website before coming back in
FY2013 (October 2012).
Those who previously predicted VB movements probably have to rework their
posts one way or the other, in a "significant" way, based on this VB.
Talking about FY2008, there are 11 more months to go through it (from
01NOV07 to 30SEPT08). Assuming the average I-485 inflow is 2k/mo. then there
would be 22k (2k x 11) NEW visa demands by the end of September 2008. Of
course you can argue that there is a (much) smaller inflow for EB2C since
the middle of 2008 but remember that I took "the average" for the simplicity
of calculation.
It seems to me that SOFAD is a better argument than any other data available
, including PERM and monthly demand data. The average SOFAD in past years is
out there and shall stay w/ a 10%+/- fluctuation range/rate. I wish you
agree; if not, your inputs are welcome.
Not sure if FY2012's SOFAD is big enough to cover that much (22k + PWMB +
inflow-of-two-recent-VBs). Again, assuming 2k/mo. inflow holds true then two
recent VBs would generate a demand of 13k AOS (2k x 6.5). Roughly for
FY2012 there would be a total demand of 35k. This figure would be very tricky for
a SOFAD to match as it probably have to stretch a little bit to reach that far, given FY2012's economy is still in the "bad" side. I would like to explore your thoughts.
Based on those PDs reportedly greened from this website I'm positive that
non-PWMB folks are pretty much done. I may missed some threads but the
latest greened PD I've ever heard was PD 13JUL07. (I noticed that there was
a person w/ PD 26JUL07 reported greened in October but his/her message holds
no accountability since that PD was not "current" yet by that time--I
considered that as a simple typo.)
Rocketsfan and Helsinki, including a few others, are on call. :p
Disclaimer: I'm a newbie here. Don't take my words seriously and don't make
your decision based on them, either. If you don't agree w/ what I said here,
at least you can leave in peace. If you would share your real thoughts w/
folks here I'm pretty sure you're welcome.
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g*e
2
【 以下文字转载自 Military 讨论区 】
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S*r
3
好多新片都有啊
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w*n
4
本人最近刚开始学作JurkatT细胞的转染.网上有说拿电转的,也有说拿Fugene6转的,请
问有哪位大虾有经验做过转染JurkatT cell,麻烦贴个protocol.多谢了.
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r*n
5
My gut feeling is that all 2008 PD has a chance of submitting 485 in the
next 3 months.
It may reach the end of 2008 in the coming 3 VBs. But of course Mr.Co can
change his mind at any time and may only advance several months.

those
after
in

【在 e*2 的大作中提到】
: I never tried to predict a VB but, as a freshman here, I did do some
: homework to try to understand VB and its trend.
: With that being said, the next VB is doomed meaningful, especially for those
: PDs in the second half of 2008 (calendar year) and later, as this VB, after
: 6.5 months' leap forward, should give us some decision power to keep
: looking forward or simply turn away from this website before coming back in
: FY2013 (October 2012).
: Those who previously predicted VB movements probably have to rework their
: posts one way or the other, in a "significant" way, based on this VB.
: Talking about FY2008, there are 11 more months to go through it (from

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r*y
6
是啊,七月十二以后,国产片都上youtube了

【在 S*********r 的大作中提到】
: 好多新片都有啊
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w*n
7
请大虾指教~
5个包子
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m*o
8
nice discussion
keep up the good work
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F*y
9
越发觉得下载没有用处了,家里的blue ray可以上YouTube直接看,爽
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d*y
10
electroporation: 1.3 x 107 cells with 10 μg DNA in serum free medium for 10
min and then electroporated the cells using settings of 300
V, 800 μF, and 24 ohms. works well, cheap.
or use amaxa nucleofection. works extremely well. a little bit expensive.
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N*r
11
No way. It could closer to end of FY12: e.g. June or July, but no way he
exceeds the FY12 to FY13. It is crazy....
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x*h
12
blue ray跟youtube有啥关系

【在 F********y 的大作中提到】
: 越发觉得下载没有用处了,家里的blue ray可以上YouTube直接看,爽
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w*n
13
在把1.3X107细胞和DNA混合以后是在冰上放10min? 电击完了以后是在室温放10min吗?

10

【在 d***y 的大作中提到】
: electroporation: 1.3 x 107 cells with 10 μg DNA in serum free medium for 10
: min and then electroporated the cells using settings of 300
: V, 800 μF, and 24 ohms. works well, cheap.
: or use amaxa nucleofection. works extremely well. a little bit expensive.

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l*e
14
为何我看youtube上的中文电影总有一顿一顿的感觉,不是很流畅。
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d*y
15

RT, 1.3X107cell+10ug DNA in 300ul Serum free medium, RT 10 min.
电击完了以后是在室温放10min吗?
Put cells in medium (10ml) immediately after electroporation.
BTW: use 4mm gap cuvettes for electroporation

【在 w*********n 的大作中提到】
: 在把1.3X107细胞和DNA混合以后是在冰上放10min? 电击完了以后是在室温放10min吗?
:
: 10

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F*y
16
blue ray的机器上有YouTube app

【在 x***h 的大作中提到】
: blue ray跟youtube有啥关系
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I*y
17
记得我以前就用Lipofectamine转,work的还不错,以前实验室有个特殊的protocol,我
现在都忘了

【在 w*********n 的大作中提到】
: 本人最近刚开始学作JurkatT细胞的转染.网上有说拿电转的,也有说拿Fugene6转的,请
: 问有哪位大虾有经验做过转染JurkatT cell,麻烦贴个protocol.多谢了.

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t*r
18
这个可以有

【在 F********y 的大作中提到】
: blue ray的机器上有YouTube app
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w*n
19
dicty 大虾, 今天我按你的protocol试了,还是没转进去。我怀疑我的细胞有问题或者
我的电击仪没设置好。
请问你平时怎么养Jurkat T的,
另外请帮我看一下电击仪的连接设置,多谢!
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w*n
20


【在 w*********n 的大作中提到】
: dicty 大虾, 今天我按你的protocol试了,还是没转进去。我怀疑我的细胞有问题或者
: 我的电击仪没设置好。
: 请问你平时怎么养Jurkat T的,
: 另外请帮我看一下电击仪的连接设置,多谢!

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w*n
21


【在 w*********n 的大作中提到】

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w*n
22


【在 w*********n 的大作中提到】

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w*n
23


【在 w*********n 的大作中提到】

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T*u
24
我也有同样的问题,电转化学转都试过了,都没work。

【在 w*********n 的大作中提到】
: dicty 大虾, 今天我按你的protocol试了,还是没转进去。我怀疑我的细胞有问题或者
: 我的电击仪没设置好。
: 请问你平时怎么养Jurkat T的,
: 另外请帮我看一下电击仪的连接设置,多谢!

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d*y
25
就是RPMI+10%FBS养的。不过可能细胞的克隆不一样,会有点差别。我们是一个个试,
试出这个条件的。
我看不懂你的电击仪的连接设置,跟我们的不一样,我们是BTX-ECM600。
实在不行,就用amaxa nucleofection吧,不过如果你们试验室没有那个仪器,得问问
别的试验室有没有可以借。
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