w*********t
发帖数: 928 | 1 bought 2x2G mem for $33
using that $10 off
there is nothing good in the 48 hr deal. |
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M********y
发帖数: 1964 | 3 前几天跳了ASUS的460，结果今天收到newegg的email给了一堆coupon，
头一个就是号称48hr(到明天中午过期)的Any SSD 20% off，俺试了一下，
咋不能用呢。 |
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B******o
发帖数: 496 | 5 第一次转染的时候用的reporter plasmid，reseed时候所有well里已经转染的细胞数是
一致的。第二次转染的时候再转你的transcription factor，即使它会促进细胞增殖，
我觉得不会对结果有多大影响，毕竟你的TF如果activate luciferase expression，差
别那是相当大(suppose你24 or 48hr收样品).细胞增殖的过程实际是你的vector在细胞
里被稀释过程，按照你的说法control background luciferase是应该降低的，如果在
这种情况下你能看到明显的activation，那一定是真的对吧
normalization可以用蛋白浓度(前提是上一样的量),和用上样的总蛋白量其实是一样的
道理
只是俺的拙见 |
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c****g
发帖数: 93 | 6 我做的是CHO cells， >10个细胞的一个克隆用我的方法基本能看到。一般的细胞及时
体积小点也应该是这个数量级吧。最后有没有clone还是依赖于你的效率，实在没有的
话就转染的plasmids多一些吧，我们一般10k cells/100mm dish，1-2ug plasmid，
48hr之后 add selection pressure。如果有clone，你后续的还要通过免疫组化或
western blot鉴定呢，通常positive的挺多的。 |
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m**********d
发帖数: 137 | 7 之前做过很多lentivirus，都是超速离心virus之后infect细胞，transduction效率都
还不错，这次偷懒没离心，直接用的含有病毒的medium，可是puromycin select一两天
后细胞全死光了，以下是详细的protocol:
vector用的是pBABE-Luc （基于pBABE-puro,只不过也表达luciferase）
293T细胞，养在75cm2 flask里，用lipofectamine2000转染的，包装病毒用的是second
generation packaging system (PAX2,pMD2.G)
分别于转染后48hr和72hr collect medium，过了一下0.45uM的filter，又加了8ug/ml
的polybrene，就加到要transduce的细胞里去了，24hr之后开始puromycin selection
我能想到的问题可能是准备transduce的细胞有点太confluent了，但这也没法解释全死
光的结果，我想很可能是没有病毒包装，高人们帮忙看看这过程中可能是么出了问题 |
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n********k
发帖数: 2818 | 8 well,it is a problem-solving issue: there are always many choices and I
would think it over and over like with any complex experimentations: what
are the options and what might be a best one to approach this...As far as I
am thinking, the less passaging the better...otherwise one might be running
a risk of picking the cells from a same clone multiple times; as well, the
clonal growth/death bias over the passaging could also dramatically change
the properties of the culture over time...numerous ... 阅读全帖 |
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k********u
发帖数: 7 | 9 i am trying to see the cell cycle phase distribution.......really don't know
what i should use, transcient transfecion for 48hrs or stable clones for 2
weeks.... |
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b****s
发帖数: 148 | 10 increase MOI
try harvest protein as early as possible, eg. 48hrs post-puromycin selection
. |
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m***m
发帖数: 58 | 11 Re!
48hrs treatment, it might have affected the transcription/translation of
actin or protein degradation in general. Actin is not always stable. I
used to study transcription factors and actin fluctuates a lot. For your
purpose of loading control, I would try all three of them. Tubulin might be
the most stable one. |
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g*********3
发帖数: 177 | 12 这个还真没有关注过，一般在70%左右transfect细胞，然后48hr后收细胞，这时候细胞
已经非常confluent了。不知道有没可能蛋白被细胞讲解。 |
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r******k
发帖数: 446 | 13 我现在想问下，如果必须做overexpression的 蛋白的half life。 是不是在
transfect 24hr或者48hr后加CHX比较好？？ 我看了一些paper，加了CHX后的time
course 时间很不一样，像我这种case，您觉得应该多久比较好呢？
endogenouse |
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r******k
发帖数: 446 | 14 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 |
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r******k
发帖数: 446 | 15 如果 transfection后 24 36 还是48hr后看signaling最好？ |
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u*****0
发帖数: 156 | 16 People should watch for social etiquette. No questions about it and no
excuses about it. I am just saying whining wont do any good. Use your
brains to smooth out the kinks. There might be better ways. Like automatic
reconfirmation 48hrs prior to the meeting date, etc. And committee完全是义
务付出. So beat it.
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k**e
发帖数: 2728 | 17 ☆─────────────────────────────────────☆
zbbh (麦地哲别) 于 (Tue Jan 4 22:49:09 2011, 美东) 提到:
有赖众多兄弟姐妹的支持，ACAP SRF自成立以来已经组织了不少次的活动，尽管疏漏不
少，但是还是受到了兄弟姐妹以及ACAP board members的肯定。但是由于我们这个群体
的特殊性（一句话概括是咱们贡献不多，要求不少，详见后续ACAP SRF小史），还是有
必要对咱们活动中暴露出的问题给大家提个醒。
在12月18号刚刚结束的ACAP年终晚会后，有不少Board members对CMG流露出失望和抱怨
的情绪，觉得我们对他们的付出不够尊重。主要原因是有好几个兄弟姐妹报了名但是没
来，也没有通知ACAP，有一个是报了一家4口，结果都没来。另外有几个人没有报名就
去参加，让ACAP Administrator Alex很被动。今年是ACAP和另一个华人医生组织CAMS
合办的晚会，有近一千人参加，组织工作量很大，花得力气、金钱都不少。ACAP也一直
希望节省开支，但是只要你报了名没cancel，座... 阅读全帖 |
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l********y
发帖数: 2295 | 18 【 以下文字转载自 Pre_Resident_Club 俱乐部 】
发信人: lovelypony (pony), 信区: Pre_Resident_Club
标 题: Tips for IM interns
发信站: BBS 未名空间站 (Sun May 27 09:34:24 2012, 美东)
INTERNS: daily management (orders, consults, pages, calls); replete lytes
daily before rounds (check for renal failure first; if Cr up, run by
resident before ordering repletion); the groundwork to get stuff done and
care for patients
--> BEFORE ROUNDS: arrive by 6am (weekdays), get signout, review all labs
and test results, see all your patients... 阅读全帖 |
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l*********3
发帖数: 190 | 19 GXGX
I took it today and I felt the same way as you were.I got 10-12 "select the
all apply" questions. It is difficult to wait these 48hrs.
Praying... |
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l******0
发帖数: 1362 | 20 you sure I was right?
Thanks for your best wish.
I have to wait at least 48hrs to know.---------it is torture!!! |
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c********e
发帖数: 42175 | 21 不是48hr之后就不扣了么？
难道不适用于旧文章？。。。版里没钱了，以后把几年前的包子全部收回来。 |
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g*****d
发帖数: 991 | 22 Antivirus med, already passed the time window, should be used in 24-48hrs. |
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w*******y
发帖数: 60932 | 24 I picked up Dragon Age Origins Ultimate Edition (xbox 360) 14.98 @ Target B&
M today. PS3 version was still $29.98.
sku is: 207220545
These Target clearance posts are kind of a joke lately as most stores sell
out very fast. Also, the employees told me the online inventory only
updates every 24-48hrs. Good luck if you can find it.
***I haven't played this yet but gamespot gave the xbox 360 version only 8.5
vs 9.5 for PC/ps3
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w*******y
发帖数: 60932 | 27 Just ran across a free small pizza from Hungry Howie's.
You must go to their facebook page and like it to receive the info for a
free coupon. Mine is good up to 48hrs from time received. Good luck all
Link:
http://www.facebook.com/hungryhowies
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w*******y
发帖数: 60932 | 28 Link:
https://www.plumdistrict.com/deals/ecomom-com-25-for-50-worth-of-all-natural
-organic-products-at-ecomom-com-UJOauf/buy_deal
$25 - $10 coupon (enjoy10)= $15
Now Ecomom is having a black friday sale (through Monday) where you spend $
50 and you'll get $20 off, spend $100 and get $40 off plus free shipping by
spending at least $50.
Since the plumdistrict voucher isnt immediately available (takes 48hrs?) it'
s unknown if these will combine.
The possibility is to buy $70 worth from your origin... 阅读全帖 |
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