v***a
发帖数: 1242 | 1 我用的是promega的pCI-neo Mammalian Expression Vector作cloning，它的manual上
明确说了insert需要有ATG来作translation initiation的，敢问各位大虾，我需不需
要在antisense primer的近5'端加stop codon呢？此vector在multiple cloning
region后是有ployA的。
谢谢您的回答。 |
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v***a
发帖数: 1242 | 2 谢谢。其实我现在的问题就是不知道该如何设计出clone全长coding sequence的primer
，因为sequence 1和sequence 2的cds也不尽相同，而且sequence 1只是partial cds，
无stop codon，而我针对sequence 2设计的primer又总是clone不到我要的gene（其实
我觉得sequence 1可信度较高，所以现在怀疑是不是sequence 2是不确切的）。我现在
想做的就是你的思路，但为难在primer上了。
PCR |
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b******r
发帖数: 111 | 3 Anyone has experience of inserting 9kb in a 8.4kb vector? 2 months was gone
,but I get nothing. Would
you like to take a look a this protocol and give me some suggestion ?
Thanks a lot
Purpose: Insert 9kb mouse sequence into vector pPNT6.
Vector pPNT6: Its map is at http://www.addgene.org/pgvec1?
f=c&identifier=11072&atqx=pnt6&cmd=findpl . I have inserted 0.9kb DNA in
this vector at XhoI/EcorI
double sites. I should have no problem in cloning short fragment.
Insert DNA is 9kb.I have cloned t... 阅读全帖 |
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a******n
发帖数: 167 | 4 Use 1 specific primer and 1 degenerate primer to do PCR then TOPO-clone.
Btw, try UCSC if you are cloning human/mouse genes. |
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l****n
发帖数: 3 | 5 I tried to study the function of a protein in cancer cells. And I
established both stable clones and a stable pool that overexpress this
protein. But I observed different phenotypes in stable clones and the stable
pool. Any explanations? Which one should I trust? |
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C*****h
发帖数: 926 | 6 不要用Topo-TA cloning。
这是我用过的，世界上最差的cloning kit。 |
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s********8
发帖数: 619 | 7 最近试了一下phusion+topo cloning,我也是PCR纯化后加taq 20分钟,但是连不上.
我理解如果PCR纯化过了再加A,就不用再纯化了.
如果PCR完了直接加A,马上topo clone,应该也可以 (according to topo manual),但是我没试过,哪位试过的同学来说说? |
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m*********n
发帖数: 158 | 8 vector 就5kb, insert 0.5kb
用pfu， pcr mutagenesis
dpni消化
然后transformation
挑clone后，我一搬习惯先跑交，发现总有很多clone size around 3kb
我vecotr 5kb也不大呀，这是为什么？
谢谢 |
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l**********1
发帖数: 5204 | 9 RE:
not expensive blunt TOPO
but alternatively you can try
another ligation-anchored PCR protocol:
details just go to
http://www.mitbbs.com/article_t/Biology/31517723.html
2F
from our experience even over 7.5kbp with a very longer 3' UTR mRNA (for
alternative splicing functions
possible) can be full cloned its ORF plus 5' UTR and near 3' UTR (3kbp)
then TA sub cloning
with TOPO TA kit:
htp://products.invitrogen.com/ivgn/product/K450001
Taq |
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r****r
发帖数: 36 | 10 1. It depends on what genes were in your plasmids. for example, it is ok to
manipulate some DNA viruses with large genome using conventional cloning
method but when it comes to some RNA viruses, you can't even clone its
fragments more than 3K.
2. I have no experience on Cosmid/YAC/BAC.
I |
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l******o
发帖数: 103 | 11 Thank you all for the kind reply.
1. I did not check the whole 4kb cDNA after site-direct mutagenesis. I sent
it for sequencing yesterday.
2. After screening positive colonies by PCR, I midi-prep DNA and transfect
into mammalian cells. After 24h, cells were harvested and directly boiled
with sample buffer. I run SDS-PAGE and probed the membrane with Ab against
the epitope. The original clone got good expression.
3. I cut the original clone with XbaI (see the following agarose gel photo).
The 4kb... 阅读全帖 |
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n****y
发帖数: 819 | 12 需要一个很简单的 cloning plasmid， 必须要有 KpnI 和 XhoI， 不能有 AvrII。 只
是用来做cloning 的， 不需要表达， 所以其他elements越少越好， 而且千万不能有
sv40 early promoter and origin， 因为里面有一个酶切位点之后会用到。 谢谢 |
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H*******i
发帖数: 196 | 14 另外随手下了个这个质粒GB文件，如果没错的话
aacagaaggCTCGAGgtaaccGGATCCtgatcaGAATTCaagggg
依次是XhoI BhamHI EcoRI 用BhamHI看着没啥问题啊？
如果要用XhoI 和EcoRI
首先你需要找一个载体 就当是PUC+Kan/clora这种
没有XhoI EcoRI MluI
然后把pGIPZXhoI到MluI clone到你的工作载体上，再用XhoI EcoRI插入目标序列，最
后用XhoI MluI切回你的pGIPZ 以后所有的clone都在工作载体上，所以一直用下去每次
也就多花3天/4天时间。 |
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D**********8
发帖数: 136 | 15 在stable clone中的试验结果在transient中无法重复
这个大家遇到过么
应该怎么理解呢？
真的有所谓long term /short term effect么？
是果断放弃stable clone 呢还是应该进一步研究呢
纠结。。。
多谢各位高手指点 |
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I***a
发帖数: 13467 | 16 你这个stable clone是怎么做的？
最近也在做一个stable clone，
但是得不到。 |
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w*e
发帖数: 740 | 17 接着这个问个问题
现在life technology也有geneart seamless cloning kit，clontech公司也有
infusion cloning kit，我个人感觉本质上这些kits和NEB gbison kits是一样的
不知道版上面有没有人比较过这几个，那个更稳定
. |
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h*********g
发帖数: 18 | 18 I got several plasmids from other lab and need to clone them and then do
maxiprep.
We have TOP10F' E.coli.
I will add 2 ul plasmid (100ng/ul) into one tube of E.coli TOP10F', mix, put
one ice for 5-30 min. Then heat-shock for 1 min at 42 degree.
Transfer the tube on ice. Add 250 ul room temperature SOC medium. shake the
tube at 37 for 1 hour.
Then spread 10-50ul onto a plate.
Is that right? I am new to cloning. Please help. Thanks. |
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c*******e
发帖数: 5818 | 19 我能不能直接用PCR product为entry DNA做LR cloning，我的target vector has R4--
ccdB--R3，我想用PCR product has L4--PCR--L3，不知这样可以不。这样可以跳过BP
cloning。多谢 |
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T*****e
发帖数: 247 | 20 看你的实验，你认为PCR没有问题，对吧。这种情况我可能会拿PCR产物测个序。确保确
实是你认为的序列。
TA cloning用的kit？ PCR结束之后怎么加的overhang？放了多久？有没有跑胶？哪一
步跑的胶？转化用的什么competent cell？
我的常规做法：
Q5扩增， 跑胶确定大小，胶纯化片段，用taq加3' overhang（PCR machine上72C
10min，然后4C，或者直接拿出来放冰上），然后立即做TA cloning （invitrogen的
kit），下面就是常规转化了
根据你的描述，我不知道你TA克隆前PCR产物放了多久，有没有过胶，有没有冻。我们
实验室一哥们说这些都会让overhang掉下来，我不知道是否真的如此。不过我这一套流
程从来没有失败过（如果competent cells没有问题的话） |
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t********9
发帖数: 536 | 21 请教大家在做Clone的时候， 做完Ligation之后，要先走胶检查是否Insert和Vector连
起来了吗，还是直接做Transformation，之后挑单Clone，摇菌，提取Plasmid，测序？
多谢！ |
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j****n
发帖数: 3370 | 22 看开啥package了
phd几年下来 没clone几千个 至少也clone了一千多个 |
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a***n
发帖数: 578 | 23 【 以下文字转载自 WorldNews 讨论区,原文如下 】
发信人: madman (NaN), 信区: WorldNews
标 题: Re: First human cloned
发信站: The unknown SPACE (Fri Dec 27 16:02:18 2002), 转信
Evidence was not presented yet, will be available.
The baby girl was names "Eve."
Four more clones are coming in weeks.
The sect Raelians are followers of Rael, a former race car driver, who went
abord an alien ship and was entertained by voluptuous female robots (is this
an oxymoron or not?).
The group of scientists gave "Not that you would, but that |
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y***n
发帖数: 99 | 24 Go to Amazon.com
Choose book
search "molecular cloning" in the tab
The 6th choice is :
Molecular Cloning : A Laboratory Manual (3 Volume Set) ~ Usually ships in 24 hours
T. Maniatis, et al / Plastic Comb / Published 1989
Our Price: $140.00
Average Customer Review:
Read more about this title... |
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P*****s
发帖数: 375 | 25 well, I'm a newer at bichemistry, so just discuss this rather than give
an answer.
I think they just define endonuleases as those -ases that can cleave
DNA at target sites. This is a must for the insertion of intron. So
there seems no way to clone gene without it. (Or you may ask: Can a
gene be cloned without cleaving DNA?) |
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c*****t
发帖数: 1879 | 26 PCR, then use a blunt-end or TA cloning kit (depending on the DNA
polymerase used) to clone it into a plasmid.
To check, pick a colony, stick it to a PCR tube w/ appropriet mix,
use regular PCR cycle w/ slightly longer 95C at beginning to break
up the cells.
Absolutely no endonuclease involved. If using pfu, at most exonuclease
activity is observed.
hoho |
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c*****t
发帖数: 1879 | 27 I asked a microbiol professor. He gave me a possibility:
if you want to clone an antibiotic resistance gene, it can be
done by increasing the dosage of antibiotics, for instance, cam,
in order to clone (ie having more than 1 copy and on a plasmid).
Only cells w/ plasmids that can produce high copy can survive.
coco |
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a****y
发帖数: 1035 | 28 Cloning pioneer Dolly the sheep put to death
By Associated Press, 02/14/03
http://www.boston.com/news/daily/14/dolly_dies.htm
LONDON — Dolly the sheep, the world's first mammal cloned from an adult, was
euthanized well short of her normal lifespan after being diagnosed with
progressive lung disease, her creators said Friday.
The decision to end the life of 6-year-old Dolly was made after a veterinary
examination confirmed the lung disease, a statement from the Roslin Institute
said.
Researchers |
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k**o
发帖数: 15334 | 31 吾朝太祖皇帝遗体还健在，可能千年不腐，以后技术强大了，随便clone出来几个也是
有可能的吧。 |
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N****g
发帖数: 5172 | 33 ☆─────────────────────────────────────☆
cmv (cmv) 于 (Fri Jul 24 15:25:21 2009, 美东) 提到:
☆─────────────────────────────────────☆
cmv (cmv) 于 (Fri Jul 24 21:03:26 2009, 美东) 提到:
up
☆─────────────────────────────────────☆
jaydige (一壶酒，两泡尿) 于 (Fri Jul 24 21:09:12 2009, 美东) 提到:
你要多少 哪个牌子的 pm
☆─────────────────────────────────────☆
NewEgg (心慈手软) 于 (Sat Jul 25 02:08:39 2009, 美东) 提到:
iphone clone是what。。。
☆─────────────────────────────────────☆
techlover (techlover) 于 (Sat Jul |
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c********t
发帖数: 5706 | 34 因为如果不clone, 调用他的上一层操作 add(0,bottom)就会在同一个list上操作，map
里的list也就变了。
stack. |
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w********p
发帖数: 948 | 35 又对比了一遍code, 大概知道问题出在哪里了。
之所以有结果如下：
Box(1,7,4)
Box(4,9,6)
Box(2,10,16)
是因为 Box(2,10,16) 比Box(1,7,4)大，所以 Box(2,10,16) 把 Box(1,7,4)的
stackmap都加上了。
if (bottom != null) {
//问题在于这句，加上就ok。
max_stack = new ArrayList(max_stack);
max_stack.add(0, bottom);
}
stack_map.put(bottom, max_stack);
return max_stack;
max_stack = new ArrayList(max_stack); 貌似是 max_stack和原答案clon
()的道理是一样的。自我拷贝下，改改地址， 挂在map上，防止上个level运算对其修
改。
书上出错，是挂到Map上以后再clone， 这个map就出错了。
再次谢谢coldk... 阅读全帖 |
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c********t
发帖数: 5706 | 36 明白了。我一般凡是要clone的，都写在修改之前。 |
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h****g
发帖数: 105 | 37 刚做了下 Graph Clone, Output Limit Exceeded. 查了半天也没看出来哪里有问题，
附上代码，请大牛门挑挑毛病，谢拉！
UndirectedGraphNode *cloneGraph(UndirectedGraphNode *node) {
// Note: The Solution object is instantiated only once and is reused
by each test case.
if (!node) return NULL;
UndirectedGraphNode * nodecopy;
queue que;
unordered_map map;
nodecopy=new UndirectedGraphNode(node->label);
que.push(n... 阅读全帖 |
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d****n
发帖数: 233 | 38 把
for(auto neighbor: node->neighbors){
graphCopy->neighbors.push_back(DFS(node,mp));
}
改成
for(auto neighbor: node->neighbors){
graphCopy->neighbors.push_back(DFS(neighbor,mp));
}
另外，可以参考我的blog for the two ways to solve this problem:
http://codeanytime.blogspot.com/2014/11/clone-graph.html |
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a**********0
发帖数: 422 | 39 如果是shallow clone 一个新obj 是不是比用constructor新产生一个 obj更快呢 |
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t****t
发帖数: 540 | 40 要升级硬盘。把c盘clone（norton ghost 15）到新硬盘。把新硬盘换进电脑启动之后
，硬盘就变成F盘的。然后什么可执行文件都不能执行。都说path错误。
都不知道怎么办？ |
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a*******m
发帖数: 127 | 41 从公园里剪植物的茎（stem）回家种（clone）合法么？在youtube上看了一下好grow
from
cutting很容易。 |
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b******o
发帖数: 5644 | 42 想要的手枪基本上置办齐了，现在看看长的。AK在NJ非法。眼力不好，得上镜子，SKS
略过。所以就在AR里选。Colt在NJ也非法，所以都是Clone。用途就是纸上敲敲洞。
AR大多还都是气导，比较脏。现在出了一些活塞式的，Sig只有556 SCM，不是一般的丑
。有没有人有Ruger SR556或S&W MP15 PSX的经验？ |
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D**********s
发帖数: 320 | 43 Star Wars II Attack of the clones
在Qui-Gon死后10年，星系共和国再次面临危机。日益壮大的分裂势力已经对共和国构成
重要威胁，为了对付分裂势力，参议院要召开一次紧急会议，对是否组建一只共和国的
军队进行表决。
此时Padme Amidala已经是代表Naboo参议院，此次前往Courasant参加表决至关重要。当
Padme的飞船刚刚降落在Courasant的时候，发生了猛烈的爆炸。幸好Padme乘坐的随行的
飞船，被炸死的是她的替身。
Jedi council和supreme chancellor Palpatine都对Padme的安全表示担心。Palpatine建
议由Obi-wan亲自保护Padme的安全。此时Anakin已经跟随Obi-wan十年，成长为一名英俊
强大的Jedi武士。再次见到Padme点燃了Anakin内心对她深藏已久的爱情。但是Jedi武士
是不允许恋爱，而Padme又是参议员，这使得两个人虽然互相倾慕，但又无法成为一对真正
的恋人。
Jedi council对刺杀Padme的事件展开调查。Padme认为是Count D |
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P****A
发帖数: 55 | 47 【 以下文字转载自 JobHunting 讨论区,原文如下 】
发信人: phdusa (出国迷), 信区: JobHunting
标 题: Postdoctoral Position - characterization and cloning of genes
发信站: The unknown SPACE (Sun Mar 25 09:41:04 2001) WWW-POST
Postdoctoral position posted on the postdoc jobs website:
http://www.post-docs.com |
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r****m
发帖数: 1264 | 48 laoniu is so popular. thus someone cloned another one. or
just another coincidence?? |
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