j****n
发帖数: 3370 | 1 直接插入两个cassette 也不是很难
就是promoter1-gene1-terminator1-promoter2-gene2-terminator2
利用酵母自身homologous recombination克隆 很容易的
我们实验室经常一次性搞个5-10个cassette 也没太大问题
★ 发自iPhone App: ChineseWeb 7.8 |
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k****s
发帖数: 1209 | 2 原文在
http://www.skizit.biz/2013/07/21/ecdysone-insect-hormone-an-ind
Ecdysone Insect Hormone Switches on Disease
21 Jul 2013 | ECDYSONE
MORGELLONS:
INSECT HORMONE ECDYSONE TRIGGERS GENETIC CHANGES
TO BUILD NEW HAIR, SKIN, NAILS and CAUSE DISEASES
SkizitGesture-2013
Morgellons is a military grade entomological terror weapon being used to
torture and kill innocent civilians. The medical community has not been
informed about this disease and its complexity keeps it from being properly
identified, ... 阅读全帖 |
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m*********D
发帖数: 1727 | 3 十年不作这个了，看还记得不记得。
作stable cell line,要单拷贝，你不能切plasmid，要保持plasmid的环。但你不能保
证每个插入的位置（on plasmid），因此插入的位置可能正好在你的表达cassette中间
，所以，要花大量时间来挑选你要的细胞系（PCR应该可以证实）。linearized
plasmid才会有多copy问题。
另外，就是stable cell line的silence问题。你的表达基因会随culture时间而
silence。这个当时是在cassette两边加特定的sequence来防止的（我连名字度不既得
利益了。）。现在不知道是不是有新技术。
另外，我们以前是用bicistronic系统，就是selection marker和你要表达的基因在同
一个mRNA上，中间有一个ribosome进入的IRES sequence,保证两个蛋白质的
translation。这个的好处是能在selection条件下成活的细胞系，表达你的基因的可能
性也大多了。你可以参见clontec的系统。 |
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h*****G
发帖数: 113 | 4 只要是mamalian codon optimized的Cas9就可以，最好带GFP or antibiotic
selection，这样可以sort transfected的细胞。
Cas9-2A-GFP/Puro-U6/H1-gRNA的最好。
对于knock-in cassette, 如果你只是single or a few nucleotide mutation, 那么
最方便的应该是直接order single stranded DNA, 长度 ～ 100 nt. Homology arms >
40 nt 就足够了。
如果是要knockin比较大的insert，比如说GFP reporter，那么可以需要克隆到质粒上,
homology arm > 500 bp，就可以了。可以有antibiotic selection，也可以不用，取
决于你的效率。 效率低的情况用antibiotic selection，之后还需要一个step，用cre
-loxp把antibiotic selection cassette给去掉，否则在很多情况下会影响你gene的表
达。
在两种情况都要... 阅读全帖 |
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发帖数: 1 | 5 有没有相关的文献呢 昨天跟人讨论从其他实验室拿到的细胞，怀疑这个实验室编辑的
细胞没有去掉selection cassette，同事说如果没有去掉selection cassette，即使它
在intron也会终止pre-mRNA的合成，最终导致带有突变的那个拷贝不能翻译成蛋白质 |
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m*********D
发帖数: 1727 | 6 对BP系统不熟悉。你上次说可以不留下任何痕迹，那肯定比Cre/LoxP好。LoxP我记得蛮
长的，就是在initron里，也担心影响splicing。
个人觉得没必要两个颜色的selection maker。这样导致在两个homologous arms之间的
insertion太大。看了他用PCR来确认正确克隆，实际上是两个HDR arm上一边一个PCR来
确认的。不知道是不是担心PCR有不确定性，还用了southern来证明integration site
。这个太麻烦。从他用一个颜色的selection marker（Fig2)来看，拿到double-
replacement的效率不错，40%。说明一个颜色就可以了。或者，我就干脆用一个G418。
我记得一个G418的表达cassette只有1。2Kb,在这个cassette两边加PB,PB外面是HDR的
arms,一边1kb。这样，要确认正确克隆的时候，用一对在HDR template外面的PCR
primers,就可以解决了。这个PCR产物是1+1。2+1=3。2kb，NEB的Q5 polymerase应该可
以了。 |
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p********y
发帖数: 5141 | 7 Chain, cassette, chainring depends on the type of your chain, weather condition (wet, cold?), how
well you maintain/clean it etc. I would say a couple thousand miles(2-4000?) is a
fair number.
But you will definitely know when to replace them, the chain will start to skip while riding, and the cassette crank will become sharp triangle instead of the square shape.
Brake pad also depends on mileage and weather condition, it need to be replaced much often than the other items. There is normally a m... 阅读全帖 |
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i*********5
发帖数: 19210 | 8 【 以下文字转载自 Cycling 讨论区 】
发信人: ironman2015 (1/2 ironman x3), 信区: Cycling
标 题: Bicycling's 50 Golden Rules
发信站: BBS 未名空间站 (Tue Oct 16 13:35:05 2012, 美东)
http://www.bicycling.com/training-nutrition/training-fitness/bi
Cyclists are innovators, constantly hunting for an edge. Over the last half-
century, we've tried thousands of methods to become stronger, faster, and
smarter on a bike—many of which have been discarded through the years.
These have endured.
1. To corner, enter wide and exit wide.
2. ... 阅读全帖 |
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h*d
发帖数: 19309 | 9 渠道太多了，想起这个
Mary: I had this guy leave me a voice mail at work so I called him at home
and then he e-mailed me to my Blackberry and so I texted to his cell and
then he e-mailed me to my home account and the whole thing just got out of
control. And I miss the days when you had one phone number and one answering
machine and that one answering machine has one cassette tape and that one
cassette tape either had a message from a guy or it didn't. And now you just
have to go around checking all these d... 阅读全帖 |
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h*d
发帖数: 19309 | 10 渠道太多了，想起这个
Mary: I had this guy leave me a voice mail at work so I called him at home
and then he e-mailed me to my Blackberry and so I texted to his cell and
then he e-mailed me to my home account and the whole thing just got out of
control. And I miss the days when you had one phone number and one answering
machine and that one answering machine has one cassette tape and that one
cassette tape either had a message from a guy or it didn't. And now you just
have to go around checking all these d... 阅读全帖 |
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f********m
发帖数: 8397 | 11 does it play cassette? buy a cassette converter for $10, then u r set |
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T1
发帖数: 4732 | 13 至少可以claim以下5条
3) "Published material about the alien in professional or major trade
publications or other major media, relating to the alien's work in the field
for which classification is sought. Such evidence shall include the title,
date, and author of the material, and any necessary translation."
5) "Evidence of the alien's original scientific, scholarly, artistic,
athletic, or business-related contributions of major significance in the
field."
7) "Evidence of the display of the alien's work... 阅读全帖 |
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f*********d
发帖数: 3358 | 14 http://news.cnet.com/8301-13645_3-10466722-47.html
Hifiman HM-801 vs. iPod, Zune: A sound winner?
Sure, iPods and Zunes can sound perfectly fine, but no one ever claimed they
were bona fide portable high-end audio devices. Their "good enough" sound
isn't entirely their fault: they're too small to house a battery potent
enough to power a high-quality headphone amplifier and a high-resolution 24-
bit/96kHz digital-to-analog converter.
The Hifiman High Fidelity Music Player HM-801 is the Hummer of ... 阅读全帖 |
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TN
发帖数: 1870 | 15 By the early 1970s, people in academic or research institutions had the
opportunity for single-person use of a computer system in interactive mode
for extended durations, although these systems would still have been too
expensive to be owned by a single person.
In the 1970s Hewlett Packard introduced fully BASIC programmable computers
that fit entirely on top of a desk, including a keyboard, a small one-line
display and printer. The Wang 2200 of 1973 had a full-size cathode ray tube
(CRT) and ca... 阅读全帖 |
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d*b
发帖数: 21830 | 16 说实话，20年前说这种话还有点用，现在，就美国的购买力，那千年一换的tower, US
Market越来越不重要了，自己的那market连像样点的手机公司都养不活第二家。
到头来砸的还是自己的脚，看看当年的VCR就知道了。要没季龙粉，估计美国人现在还
在用cassette. |
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s*****r
发帖数: 11545 | 17 下次该往汤水里吐痰了
The dinner bill for three friends at Chilly D's Restaurant stung, but it
wasn't the price - printed on the top of the receipt were the words "Fat
Girls".
"I got the bill, I was looking at bill [and] I was like, 'Why does this
receipt say 'fat girls?'" customer Christine Duran said.
The friends had dined out at the Stockton, Calif., restaurant, which is a
part of the Cameo Casino Restaurant, on Thursday. The bill lists charges for
three tri-tips with fries and three sodas, for a total ... 阅读全帖 |
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l*y
发帖数: 21010 | 20 Is there any good news out of China?. |
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l*y
发帖数: 21010 | 21 Even an atheist with anger issues wouldn't beat up a Tibetan Monk. |
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l*y
发帖数: 21010 | 22 This bullshit that China uses to claim territory, reaching back to the Qing
dynasty or some bullshit, needs to stop immediately. The world needs to recl
aim Tibet for Tibetans. |
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l*y
发帖数: 21010 | 23 Not to sound racist.. but every single time there's a post involving China o
r the Chinese that makes it to the front page, it is something awful.
Qing
recl |
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R**Z
发帖数: 366 | 24 well deserved. Fuck you, bitch |
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s*******u
发帖数: 9508 | 25 老秃驴还折腾啥啊, 难道不明白自己现在就是一条狗么? 让it"汪汪"两声,it就"汪汪"
两声. |
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T**********e
发帖数: 29576 | 26 “The Most Handsome Chinese Man I’ve Ever Seen”
Posted by Jiayang Fan
Fei-Xiang.jpg
http://www.newyorker.com/online/blogs/culture/2012/01/fei-xiang
In 1987, a young man walked onto a vast, lantern-festooned stage and, in
less than three minutes, won the hearts of a billion. The stage was the
Spring Festival Gala, a nationally broadcast variety show that makes the
Radio City Christmas Spectacular look homespun. But Fei Xiang, who soon
became the Gala’s main attraction, was no mere Rockette. Fei st... 阅读全帖 |
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b***y
发帖数: 14281 | 27 哈哈，这个不是一般的脑残。VCR是指的卡带式录相机，video cassette recorder. 现
在哪还有什么VCR，直接说短片得了。
★ 发自iPhone App: ChineseWeb 8.2.2 |
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f***y
发帖数: 4447 | 29 一个颜宁能顶十个千人
http://news.sciencenet.cn/htmlnews/2017/6/378809.shtm
2017年6月8日，清华大学生命学院、结构生物学高精尖创新中心颜宁研究组在《细胞》
(Cell)杂志在线发表了题为《人源脂类外向转运蛋白ABCA1的结构》（Structure of
the Human Lipid Exporter ABCA1）的研究论文，首次报道了胆固醇逆向运输过程中的
关键蛋白ABCA1近原子分辨率的冷冻电镜结构，为理解其作用机制及相关疾病致病机理
奠定了重要基础。
胆固醇广泛地存在于高等动物的各类组织细胞当中，它不仅是细胞膜、血浆脂蛋白的重
要组成部分，也是包括胆酸、维生素D、类固醇激素在内的许多特殊生物活性分子的前
体化合物。但是，人体内过量的胆固醇积累会促进血管动脉粥样硬化的发生和发展，并
有可能导致严重的心脑血管疾病（如冠心病及中风等）。正因为胆固醇对于人体健康具
有两面性，所以细胞内的胆固醇平衡(cholesterol homeostasis)对于维持人体的健康
是必须的。细胞内的胆固醇平衡涉及一系列受严格调控的过程(图1)，例... 阅读全帖 |
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k***g
发帖数: 4904 | 30 老邱，别激动，你修的变速器cassette都没法拆成一片一片的吧？呵呵 |
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M*V
发帖数: 3205 | 31 下次该往汤水里吐痰了
The dinner bill for three friends at Chilly D's Restaurant stung, but it
wasn't the price - printed on the top of the receipt were the words "Fat
Girls".
"I got the bill, I was looking at bill [and] I was like, 'Why does this
receipt say 'fat girls?'" customer Christine Duran said.
The friends had dined out at the Stockton, Calif., restaurant, which is a
part of the Cameo Casino Restaurant, on Thursday. The bill lists charges for
three tri-tips with fries and three sodas, for a total ... 阅读全帖 |
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j******w
发帖数: 4429 | 32 I am shocked。。。
MJ 是king of pop，那是西方人的king, 80年代不拿歌词 你能听懂mj唱什么？
(我们那就人唱毙咧，毙咧。。。。),我刚开始听整个一个惊声尖叫么，后来
we are the world, heal the world 等慢歌旋律还不错，。。
和我说MJ舞跳的好，确实不错，可是几个人去看过现场？和我提mtv mv 对不起 80年代
大陆就没那东西
80年代主要还是谭咏麟 张国荣 梅艳芳的，学友也不错。。。MJ 麦当娜就像麦当劳一样
吃个新鲜，（你也可能说我提着几个说不定又都是mj的粉丝，so what..只是喜欢这些
人的
歌而已，没有跟风的必要。。。）当主食肠胃受不了，这也是为什么不懂mj死了中国网
友反应这么大。
老邢的mj私生活说我就不评价了，也不是很care...
最后说说盗版和销量问题。
1。华人歌手销量最多的是歌神张学友，当然周杰伦亏在盗版太猖獗了
汗一个，我2000后就没买过cd cassette了
80年代也盗， 翻录，当年我们攒都是买很多sony空白磁带，翻录，还去复印磁带封面
搞的和真的一样，这样算下来 |
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g********2
发帖数: 6571 | 33 NEW YORK – During a recorded phone conversation with Gennifer Flowers in
December 1990, Bill Clinton can be heard reacting to a charge that he had an
affair with former Miss Arkansas Lencola Sullivan, the first African-
American woman to place among the top five in the Miss America pageant.
“I can see why, you know (inaudible) she’s darkened my door,” Clinton
told Flowers, after denying the affair during the conversation.
Listen to the audio here:
From December 1990 to December 1991, Flowers fam... 阅读全帖 |
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a***n
发帖数: 1616 | 34 Audio Car Kit.
一个假磁带样的东西，输入耳机头接你外置audio设备的耳机输出，
磁带插到car cassette player。
walmart有，记得和CD player一起卖的有20，30刀一套的，单卖有15刀一个的。
还比较好用。 |
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a***n
发帖数: 1616 | 35 嘿，让偶看看，先。
如果质量好的话，偶不如换这个得了。
不知道普通cassette插座一样不一样。。。
92-和93+的headunit的唯一差别就是一个不支持CD changer，一个支持么？ |
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b*****h
发帖数: 783 | 36 几周前刚买的2000 saab 9-5 se, 72K,突然启动时发动机抖动不已,挂挡发动机抖的更
厉害,几乎要熄火, 然后engine light亮,有汽油味,随后变成臭鸡蛋味.
engine codes 是p1300,p0300, (ignition system or misfire)
上saab 论坛查询,最有可能出问题的部件是direct ignition cassette,
dic很好拆,一个引擎前后两个,拆开前面的dic发现里面有条裂缝,多半就是他了.在
eeuroparts上买了一个225刀,还真不便宜, 装上后,车就正常了.
感谢wangq008 提供的各种有用网站信息,
saab车容易出问题,大家有机会多多交流.呵呵. |
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s******g
发帖数: 780 | 37 2007 Ford Taurus SE,
3.0 V6,
automatic,
air, tilt wheel, cruise control, power windows, power locks, power seat,
rear wing, aluminum alloy wheels, AM-FM-Cassette, 4 new tires, Arizona Beige
with Beige cloth interior,
35,000 miles with remainder of 5 year/60k power train warranty,
$8,300.
VIN # 1FAFP53U37A203721.
Two owner rental from Texas. |
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L*****y
发帖数: 4290 | 39 2001 Toyota Corolla CE 4D Sedan, 86K, automatic gear, Air Conditioning
AM/FM Stereo Dual Front Air Bags
Power Steering Cassette
4个月前换的break pads & shoes,1月前换了两个轮胎
要价 4800 是不是很贵啊 |
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d**o
发帖数: 18 | 40 我说了，这是我发布的广告：
1997 Mazda protégé LS in good condition power windows, power locks, cruise
control, stereo cassette with am/fm, 172xxx miles. The body is in fair
condition. The registration expire NOV/2010. Passed the smog test on DEC/
2009. It runs good on local/freeway. The only problem is the car vibrates a
little in idle and makes a little bit noise. Auto-repair said one of the 4
cylinders is misfire after code reading, the car will stop vibrating as soon
as replacing new spark plugs (which wi |
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g**u
发帖数: 583 | 41 新手第一次买车，还请各位牛牛发表下看法，
如果好心人帮个忙查一下这个的VIN： 2HGEJ6671WH529523，更是感激不仅，随后送上包子，聊表谢意。
车子的信息：
HONDA Civic LX 98. It has only 89,000 miles on it.
Price: $3800.
Kbb faire price is 3815
features:
Four door silver colored car with 4 cylinder and 1.6 liter engines, FWD,
automatic transmission, power windows, power exterior mirror, airbags (
driver and passenger front), cruise control, cassette player, AF-FM radio,
remote (key less entry), also equipped with theft prevention alarming system
. Safety and emiss |
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s********t
发帖数: 1429 | 42 【 以下文字转载自 NewYork 讨论区 】
发信人: sweetsweet (sweetsweet), 信区: NewYork
标 题: 卖车 2005 Audi A4 V6 3.2L S-line only 25k miles - $17600
发信站: BBS 未名空间站 (Fri May 14 15:18:15 2010, 美东)
在jersey city
可以另外购买4年的warranty $1700
刚从加州运来 一个月开不到2次 准备卖了
Sport Package
Mileage: 25,000
Engine: V6 3.2 Liter
Transmission: Tiptronic Auto
Drivetrain: Quattro AWD
Air Conditioning , Cruise Control, Front Side Air Bags
Power Steering, AM/FM Stereo, ABS (4-Wheel)
Power Windows, Cassette, Leather seats
Power Door Locks, Multi Com |
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A*****d
发帖数: 128 | 43 车是2000年黑色的，是私人的，第二任车主。
奇怪的是，carfax没有事故报告，autocheck有一个事故报告，不过autocheck的评分还
比较高，86分（这种车的平均分是73-89分）
主要配置是：
3.0L V6
Auto Climate Control
Leather Interior
Heated Front Seats
Dual Front Air Bags
Anti-Lock Braking
Power Windows
Power Door Locks
Power Steering
Tilt Wheel
Cruise Control
Traction Control
AM/FM Stereo
Cassette
Dual Power Seats
Single Compact Disc
CD Auto-Changer
Moon Roof
Alloy Wheels
Clean title history!
大家看看怎么样啊，值不值？ |
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g*****p
发帖数: 3403 | 44 Info on my 2006 Nissan Maxima SE
Mileage: 46844 miles
Year:2006
Body type: Sedan
Engine: 6 Cylinder 3.5 Liter
Exterior color: Black
Transmission: Automatic
Fuel type: Gasoline
Interior color: Gray
Drivetrain: 4 Wheel Drive
Standard equipment
Power Windows, Air Conditioning, Cassette
AM/FM Radio CD, Power Steering,
Cruise Control, Power Door, Locks Dual Zone Air Conditioning
Sunroof,
Power Seat Drive,r
Dual Air Bags
Non-Smoker Vehicle!
This Vehicle is front wheel Drive and has a moon roo |
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y***m
发帖数: 7027 | 45 1 owner
2001 Toyota RAV4
4-Door 4X4 4WD
Vehicle Engine 4-Cyl. 2.0 Liter
Automatic Transmission
Mileage 96000
Equipment
Cassette,Cruise Control,Air Conditioning,Single Compact Disc,Tilt Wheel,
Power Steering,Power Door Locks,Power Windows,Privacy Glass,Dual Front Air
Bags |
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l******k
发帖数: 19 | 46 听说Audi出了名的毛病多，俺是新手，对车一窍不通，不知道也。
看了这个车， 6500刀
* 2001 AUDI A6 QUATTRO AWD
* 111K MILES
* 2.7 LITER TURBO ENGINE AND AUTOMATIC TRANSMISSION
* TAN LEATHER INTERIOR
* FACTORY IN DASH AM/FM CASSETTE AND CD RADIO
* POWER WINDOWS, LOCKS, MIRRORS
* DUAL POWER HEATED SEATS
* DUAL CLIMATE CONTROL
* WOOD GRAIN ACCENTS
* TILT WHEEL CRUISE CONTROL
* POWER MOONROOF
有carfax报告。
谢谢了 |
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g*******8
发帖数: 9 | 47 今天下午刚找人帮忙去试驾了一辆车，2002年toyota Solara SE 2D， 99550迈，感觉
还可以。卖家给的配置如下：
Air Conditioning , Power Steering, Dual Air Bags, Power Windows
Power Door Locks, Tilt Wheel, ABS (4-Wheel), Cruise Control, AM/FM Stereo
Power Seat, Cassette, MP3 (Single Disc) , Premium Sound, Alloy Wheels.
卖家要价为$6000，我不懂车，大家感觉怎么样呀？ |
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f******9
发帖数: 28 | 48 2002 Honda Civic LX 4D Sedan in excellent condition up for sale at a throw-
away price. Free Garmin GPS. See attached pics
Expected Price: $ 5,500
Car title history: Clean
Color: Silver metallic
Mileage: 76,968
Engine: 4-Cyl, 1.7 Liter; Transmission: Automatic; Drive train: FWD
Air Conditioning,Tilt Wheel, Dual Air Bags, Power Steering, Cruise Control,
Steel Wheels, Power Windows, AM/FM Stereo, Power Door Locks, Cassette
看中这辆车，VIN是： 1HGES165X2L053543.有人有access，可否帮忙查询下这车的历史
和title是否clean。万分感谢！
ps |
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w*****y
发帖数: 736 | 49 2004 Toyota corolla LE, 89K miles, $6000. tan w/tan interior, automatic,
power locks, windows & mirrors, cruise control, CD, cassette, A/C.
We are the 2nd owners. There is absolutely nothing wrong with it, has no
leaks.We have done all the scheduled maintenance since we bought it. We have
our maintenance records, but not the previous owners. I may be able to get
them from the dealer though. We replaced the brakes & tires, but no other
major repairs. My husband is a mechanic. We have never regist |
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h*****e
发帖数: 4 | 50 2002 Chrysler Sebring LXi Sedan 4D - 95,000 miles
95,000 miles
2.7Liter V6, Automatic, FWD
Tan exterior and interior
Fully loaded with:
Dual airbags and Side airbags
Power steering, door locks, windows, and driver’s seat
AM/FM Stereo with cassette and 4 disk changer in dash
17” Alloy wheels with 4-wheel ABS
Moon Roof
Leather seats
Remote starter installed
A/C and cruise control
发现身边人没什么买克莱斯勒的，现在价钱谈到4300左右,original owner，轮胎是新
的，其他东西不知道她换过没有，如果都是original，什么部件需要注意的。望指教 |
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