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全部话题 - 话题: ligation
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s******s
发帖数: 13035
1
来自主题: Biology版 - another clone trick: 3-way ligation
I can tell u what I have done in China
Synthesis 10 ssDNA of 60Nts each (indeed, should be 5 dsDNA with
anealing end), ligate 6 together to get Mixture1, ligate the other
4 to get Mixture2, ligate Mixture1, Mixture2 with vector and finally
got the right product. It means to ligate 10 ssDNA (5 ds fragment)
with vector in one step, and it works!!!!
g***m
发帖数: 465
2
来自主题: Biology版 - Re: Ligation Problem
man, refer to gzip's post.
the ligation is somehow vulnerable.
1. check with your lab fellow to make sure your ligase is still alive.
2. always use latest ligation buffer, you can even smell the thio stuff if you
are using NEB enzyme.
3. critical thing is your insert: never directly use the insert purified from
gel, you need gel purification followed by ethanol precipitation to make sure
the purity. However, you can use gel extracted vector for ligation in small
volume.
4. add enough insert, for
T**********t
发帖数: 1604
3
有,叫splinted ligation。
因为T4 DNA ligase的功能是特异性修复双链DNA上的nick。所以需要设计一个splint
oligo,和你需要ligate的两个oligo的两端各自互补。像这样:
图里显示的是连接RNA,不过DNA也是一样的。
这个protocol在这里:
http://www.megaupload.com/?d=QT50D23R
[7] Joining of RNAs by splinted ligation
Melissa J. Moore and Charles C. Query
Methods in Enzymology
Volume 317, 2000, Pages 109-123
RNA - Ligand Interactions, Part A
C*******e
发帖数: 4348
4
hoho,我这两天做的也是基于pUC19的
是pUC19(SacI)-(SacI)fragment I(XmaI)-(XmaI)fragment II(BamHI)-(BamHI)
fragment III(XbaI)-(XbaI)fragmentIV(HindIII)-pUC19(HindIII)
每个fragment大概0.8-1.1kb
然后我做的是pUC19 50 ng,
pUC19:f1:f2:f3:f4=1:5:5:5
总体积15-20 ul
ligate 20 hrs
然后3 ul加到一管TOP10电感化态细胞里
涂100-200 ul/平板
然后挑克隆
用colony digestion筛选
一起做了五个ligation
一天筛了近90个克隆
每个ligation都筛出正确的了
然后这些positive的提质粒出来
用不同的酶切再确认一下
然后测个序(或者不测)
直接PCR的切就行
不过我个人比较喜欢的是每个片段先做到TOPO里面(pBluntII)
先测序
挑序列正确的再酶切、胶纯化,往下做
这样每一步都是正确的序列
最后终产物酶切什么都对的话不测序也没关系
不然如果... 阅读全帖
a***o
发帖数: 129
5
来自主题: Biology版 - Re: 有没有做4-piece ligation的?
Nothing special. Inserts ratio should be 1:1:1. Vector amount around ~50ng.
Inserts the more the better.
I use epicentre's fast-link ligase. I do three-way ligation routinely and
never failed once. Once for a while I do 4-way ligation, sometimes even with a
blunt end:)

stupid.
w*****n
发帖数: 107
6
the traditional T4 RNA ligase has very low ligation efficiency. While T4 DNA
ligase is very robust.
t*m
发帖数: 4414
7
hehe, you can ask guys on the "biology" board.

ligation?
l*****k
发帖数: 587
8
来自主题: Biology版 - another clone trick: 3-way ligation
Well, I am sure I am not the first to do this, but I do want to share it
Sometimes, cloning multiple things into a vector can be tedious as you
have to transfer the fragments through several vector to get the
desired one, usually because the existence of the same Restriction site
in the vector and the fragment you want to clone.
say a gene has 2 frags in two separate vector, now you wanna get
them ligated into the same vector.
EcoRI------------XhoI Frag I (has interal BamHI site)
XhoI-
v*******3
发帖数: 119
9
刚刚从生理变成了分子的人
问问
看到很多文章作了western 后southern blot之后,最上面有一个三角形,不知道什么
意思
还有什么是ligation-mediated PCR
多谢多谢
w******e
发帖数: 1187
10
要ligate two short ss oligos (~80 base for one, 10-30 base for the other),
有可能达到高效率吗(比如至少20%)?隐约记得可以用complementary sequence
作为guide来提高效率,不知有没有现成的protocol?请牛人指点。多谢!
r******y
发帖数: 21907
11
来自主题: Biology版 - ligation的问题
我的ligation死活不出东西,原来一做就出,现在折腾多次未果,昨天colony PCR出了
带结果质粒酶切后nothing,一定要在节前做出来,生物同修们,有啥建议?我订了新的
ligase,估计是buffer冻容多次就不好了?
r******y
发帖数: 21907
12
来自主题: Biology版 - ligation的问题
同样的ligation以前做过,没加过DMSO,都是按protocol做的,昨天我做了colony PCR
,就是直接拿colony做template,用我的gene specific primer扩增,有我要的大小的带
,但是提了质粒再酶切没有我做的gene的insert......
o*****r
发帖数: 156
13
来自主题: Biology版 - 如果往ligation buffer里加ATP
sometimes for old buffer, the ATP might be hydrolyzed
add additional ATP to 1 mM (final conc) would help for the ligation.
h**********r
发帖数: 671
14
用NEB的Quick Blunting Kit,把PCR产物处理一下就OK了。里面有磷酸化的功能。
载体去一下磷酸化,应该就O了。
Purchase the Quick Blunting Kit with the Quick Ligation Kit and save 15%.
目前NEB还有点小折扣。我用过,还行。
C*******e
发帖数: 4348
15
+1
我做过无数克隆了
从200bp的到12kb的insert
最近还做比较crazy的vector加4个insert片段一起ligate
从来都是vector:insert是insert过量
不管Insert长短
C*******e
发帖数: 4348
16
去磷酸化的vector加sticky ends的insert没问题
其实去不去磷酸化什么的都无所谓
我大多数情况下都不去
如果ligation的时候出问题多半不是去不去磷酸化什么的
多半是片段重新准备啊,浓一点,干净一点
多试几个ratio
vector多放点
就差不多了
vector去磷酸化不过是让自己放心
trouble shooting的时候可以排除掉一个vector自连而已
b******n
发帖数: 4225
17
用高保真酶扩,可以做blunt end ligation或者用topo之类基于isomerase连到载体上
t*****a
发帖数: 308
18
来自主题: Biology版 - 请教1 Ligation reaction question
Try this?
https://www.neb.com/protocols/2014/10/27/ligation-of-a-dna-or-rna-oligo-to-
single-stranded-rna-using-t4-rna-ligase-1

T4
a
b******r
发帖数: 111
19
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
k******g
发帖数: 1540
20
☆─────────────────────────────────────☆
orchiddream (自在呼吸) 于 (Mon Dec 17 09:30:23 2012, 美东) 提到:
LG今天教完课回家的路上经过一间Apple店.LG是电子产品爱好者,第一代ipad出来的时
候就买了一个。我对ipad, iphone这些其实不太感冒的。所以每次去苹果店都是因为LG
提议才会去。这家店是我头一次去。我们先试了一下iphone,然后又去看ipad。用着觉
得真好用啊,那么多功能。不少应用软件。然后和我LG讲买这个给我当礼物吧。LG说好
,你想要iphone还是ipad,我说我要ipad,看网页不费劲。我于是在ipad上打开网页在
那里浏览,还没有两分钟。其实就我和LG进这家店,最多也就5分钟。然后一个二十多
岁的中国男sales突然走过来和我说,may I borrow the ipad?话说着,手就伸过来拿
了。我当时也没反应过来,而且一般又是很nice的人,就跟他说,sure, go ahead. 他
旁边还站了另两个人,他要给那两个人做demo. 手里没ipad了,... 阅读全帖
R*s
发帖数: 2041
21
ft. 你们用英文是讲不清楚了, 要不就是相互都没pay attention好好看。
GENESIS的意思是不管你用多少细胞,如果你的细胞好,antibiotics质量也好,
ligation做得也clean,那你negative control就应该很clean, 没什么clonie.
跟用了多少细胞没关系。同时ligation plates里应该有大量colonies而且
false positive的比例也应该很少。所以没必要降低细胞量。也没必要因为
clonies多而多做miniprep.
除非你切了后的vector超级sticky, 而且CIP不干净,
导致self-ligation很多(这句偶加的,呵呵)。
当然偶自己做cloning很烂,经常ligation plate才长一个,
一挑还真有insert. 哈哈,倒是省miniprep.
t**********8
发帖数: 223
22
来自主题: Biology版 - 问个3c,4c的问题,包子答谢
准备做4c了,看着protocol挺简单的,就是酶切,ligation,再酶切,再ligation,但
听说很难做成功,难道有什么trick吗?有做过的能不能谈点经验和教训?
这个实验麻烦的是,感觉挺难control,一轮做下来要一两周,直到测序,除非拿到想
要的结果,否则你怎么知道中途有没有问题呢?我研究了一下,觉得3c的ligation可能
比较关键,是不是要严格控制dna的浓度和ligation的时间啊?
多谢了!
D*********t
发帖数: 370
23
来自主题: Biology版 - 跪求帮助:为什么克隆不出来
This is very difficult. I don't think I've done 4-fragment ligation (large
fragments!). You may try to
use a shuttle. E.g., put two of them into pKS first. Then ligate another
one. Then you cut out a
(big) single fragment and ligate with your vector. Someone may have
suggested this, I didn't read
all the posts.
s****a
发帖数: 436
24
最近做DNA ligation. 请问大家,DTT在T4 ligation 中的作用是什么?查了资料,DNA
ligation中主要起作用的是ATP 和 Mg2+. DTT是不是起到保护ATP,防止其被氧化的
?请问有专门的文献介绍DTT的作用么?Thanks.
s******y
发帖数: 28562
25
来自主题: Biology版 - 问个单链DNA连接的问题
FT! FT! FT!
Of course you cannot ligate the single strand DNA like this way! Because
there is NOTHING to hold these two fragments together to allow the ligase to
act.
If you really want to ligate the single strand DNA, at least you should use
some kind of carrier system like BSA or other stuffs like a non-specific DNA
-binding protein.
If you choose to ligate from the double strand DNA, you can of course break
it down to single strand by incubating them on high temperature with
denaturing reagen
n********k
发帖数: 2818
26
谢谢你的详细意见。首先要说,我做分子克隆的经验不是很多。其次,基本不会不改变
条件反复重复。
1 当初选用pQE8也是没有办法,因为pQE9我们实验室没有(pQE8和9的区别就是8在酶切
位点之间只有一个碱基,而9要长得多)。最开始的时候没有意识如此近的酶切位点很
难切下来,最起码同时酶切不可能切下来。但是只尝试过一次顺序酶切不行就没有再尝
试用pQE8了,我不记得是先切BamH1还是Hind3,后面尝试插一段片段在2个酶切位点之
间再切。
Nice alternative, shall solve the problem in this case...but I would just do
blunt ligation, nowadays, the fast ligation kits from different companies
such as Roche, takara, don't really care about whether it is sticky or blunt
ends, it works just as well...BTW, any one else ... 阅读全帖
N2
发帖数: 81
27
It is really hard to say yes or no.
Most of time, if you do just column purification, you got low ligation
efficiency because the primers in it.
it always depends specific case. For some hard ligation, you need to be
careful for every steps.
There are many reasons to fail a ligation. you may just meet a hard bone.
You can drop me a mail with the vector and inserts sequences, your primers.
I can help to have a look.

points
also
R****n
发帖数: 708
28
Generally speaking adaptor and primer are all oligo nucleotide sequences.
The difference lies in their roles in a 2nd generation sequencing reaction.
Adaptor is used for ligating the primer to the target sequences(usually for
DNA,RNA,miRs).Certain sequences are more efficient for ligation reaction.
Sometimes there will be a barcode sequences between adaptor and primer to
identify each sample when multiple samples are sequenced.common primer pair
is used for amplify a huge pool of ligated sequenc... 阅读全帖
f**********e
发帖数: 1994
29
CG 的 read length 非常短。他們 interview 我時還問我 35+35 mate pair 比較好還
是 45+25 比較好。我倒。這是什麼年代啦。
他們很便宜是因為他們不像 SOLiD 一樣傻逼要 push ligation cycle。Ligation
cycle 深下去需要的 probe 濃度會狂升(不然你讀到的就是屎),CG 每個 primer 做
兩個 ligations 拉倒。這樣子需要的 probe 很少,而 probe 是最貴的 reagent。
f**********e
发帖数: 1994
30
CG 的 read length 非常短。他們 interview 我時還問我 35+35 mate pair 比較好還
是 45+25 比較好。我倒。這是什麼年代啦。
他們很便宜是因為他們不像 SOLiD 一樣傻逼要 push ligation cycle。Ligation
cycle 深下去需要的 probe 濃度會狂升(不然你讀到的就是屎),CG 每個 primer 做
兩個 ligations 拉倒。這樣子需要的 probe 很少,而 probe 是最貴的 reagent。
s****a
发帖数: 436
31
【 以下文字转载自 Biology 讨论区 】
发信人: sevana (dreamer), 信区: Biology
标 题: 请教问题:DTT在T4 ligase, ATP中的作用是什么?
发信站: BBS 未名空间站 (Sat Apr 17 21:16:26 2010, 美东)
最近做DNA ligation. 请问大家,DTT在T4 ligation 中的作用是什么?查了资料,DNA
ligation中主要起作用的是ATP 和 Mg2+. DTT是不是起到保护ATP,防止其被氧化的
?请问有专门的文献介绍DTT的作用么?Thanks.
s**********r
发帖数: 67
32
来自主题: Chemistry版 - two paper help
1.Nina G. DOlinnaya, Marta Blumenfeld*, Irena N. Merenkova,etc.,
Oligonucleotide circularization by template-directed chemical ligation, Nucl
. Acids Res. (1993) 21 (23): 5403-5407. doi: 10.1093/nar/21.23.5403
2. Franziska Diezmann, Hendrik Eberhard, Oliver Seitz, Native chemical
ligation in the synthesis of internally modified oligonucleotide–peptide
conjugates, Biopolymers (Pept Sci) 94: 397–404, 2010.
3. K. Achilles, G.v. Kiedrowski,Kinetic model studies on the chemical
ligation of oligonucle... 阅读全帖
o******s
发帖数: 2946
33
The Chinese government isn't getting rid of its one-child policy
currently in place. It's just making it sound better. China's communist
party newspaper, People's Daily, reports that the government will revamp
its abrasive-sounding slogans surrounding the policy.
People's Daily cites several examples of "harsh slogans," including
those "which sometimes even threaten criminal acts." The newly
instituted program, slugged the "face-washing project," will offer more
proactive slogans to help enforce... 阅读全帖
o******s
发帖数: 2946
34
造谣吧
Some examples of the more offensive slogans currently in use include:
"If you don't receive the tubal ligation surgery by the deadline, your
house will be demolished!"
"We would rather scrape your womb than allow you to have a second
child!"
"Kill all your family members if you don't follow the rule!"
And…
"Once you get captured, an immediate tubal ligation will be done; Should
you escape, we'll hunt you down; If you attempt a suicide, we'll offer
you either the rope or a bottle of poison."
m*s
发帖数: 6855
35
【 以下文字转载自 Biology 讨论区 】
发信人: ligation (ligation), 信区: Biology
标 题: 生物学最大的痛苦是造假还不挣钱
发信站: BBS 未名空间站 (Mon Feb 1 04:19:43 2016, 美东)
上市公司造假数据骗散户
股市放假消息
领导层放风裁员
卖包子的放点假肉
或者造点假钞
至少还能挣挣钱,骗骗人,有点乐趣。
干生物的就是造假了也不挣钱,还跟吸了白粉似得。结果社会上有两种骂声,一种是动
物保护协会的说你不人道。还有就是临床大夫说你不挣钱还花钱,简直就是混账无赖嘛。
还有一种就是明明知道自己做的细胞培养跟真实情况没啥联系,也是刺激来刺激去,编
故事。
说白了干生物的很多就是坐牢。天天呆在没有窗户的地下室了杀夜行动物。双手双脚都
被签证身份工资至酷着。谁要是能引领大家逃出去,就是我心中的摩西,心中的毛主席。
w*******e
发帖数: 15912
36
【 以下文字转载自 Biology 讨论区 】
发信人: ligation (ligation), 信区: Biology
标 题: 为什么大家都愿意当生物千老也不愿意回国?
发信站: BBS 未名空间站 (Sun Jul 3 09:18:46 2016, 美东)
隔壁的小姑娘年级轻轻文章不少,哈工大给教授,工资保证29万一年也不回去。没有青
年千人不回,还必须解决老公。周围还有很多博士后也是老到不行,都是一轮一轮的做
。无论是气色上生活上也没见有多幸福。牛逼的帅的拍个照片上个微博。整整西餐,拍
拍老外。女的有不少拖家带口的嫁个老外,做个中餐。
现在国内大城市很多药厂,公司,高校年薪也都是20-50万,甚至更高,海外博士混混
应该很好找到职位。很好奇为什么这么多人包括我自己都不回去呢?国内的可怕之处除
了空气,食品还有什么其他的要考虑吗?高房价?如果国内房价那么高工资那么低为什
么房价还不断的涨呢?
j****e
发帖数: 919
37
来自主题: NextGeneration版 - 有没有JMs剖腹产后作了结扎的
二宝6月初预产期,预定C Section.想着宝宝生了后,反正肚皮是开着的,干脆作个tubal
ligation.就不会有老三,老四这样的意外了
问了OB,说手术很安全,就几分钟,最适合剖腹产后作,对身体也没影响.
我在网上查到有人说结扎后对身体有影响的,说是好像对激素方面的
有没有姐妹们做过tubal ligation的,能否说说看
d********f
发帖数: 43471
38
来自主题: Joke版 - 4选一,选哪个学生 (转载)
【 以下文字转载自 Biology 讨论区 】
发信人: ligation (ligation), 信区: Biology
标 题: 4选一,选哪个学生
发信站: BBS 未名空间站 (Wed Dec 12 18:52:45 2012, 美东)
4个学生,3个老外。一个是东北的中国绅士,一个是北方土著,一个是南方大家闺秀,
一个是东欧外籍。
目前在中国和北方土著中重点考虑。老外周末肯定不干活,平时还可以。老中的话英文
不行,底子差,很烂的大学毕业的小留。。。
难啊。大家给个主意,中国学生好不好?第一次选学生。。。。。
f**********r
发帖数: 18251
39
来自主题: Joke版 - 学生4选一续 (转载)
【 以下文字转载自 Biology 讨论区 】
发信人: ligation (ligation), 信区: Biology
标 题: 学生4选一续
发信站: BBS 未名空间站 (Mon Feb 25 07:15:37 2013, 美东)
上次说选学生,最后还是选了一个洋妞,选了一个中国学生。洋妞现在走了,不知道回
不回来。就单表中国学生。
这个中国学生很奇怪,不跟别人交流,平时就是跟我说中文。试验还不错,pcr,
western都不用教直接能做了,能干博士的活,但是有点让我很不爽
1.从来不谈工作以外的事情,他的生活等等一概不提。也不是我想知道,后面再说
2.我对他的要求就是周一到周五辛苦一点,周末不要来,省的坏了我的名声。但是他平
时基本11点才来,5点左右就走,有的时候比较晚。每天哈气连天的,一点精神都没有
,像抽了大烟似的。不知道怎么从300多人选出来的。我真想知道他在干神马。
3,对我的提醒视若无物。我告诉他工作得努力点,怎么也凑40个小时一个星期,别跟
我一个作息时间,我是无所谓每周20-30个小时,但是我晚上看文献也写东西啊。但是
第二天
他就12点才来,也不给个理由。... 阅读全帖
E*****m
发帖数: 25615
40
来自主题: TrustInJesus版 - Tussling Over Jesus-- NYT (墮胎爭議)
http://www.nytimes.com/2011/01/27/opinion/27kristof.html
January 26, 2011
Tussling Over Jesus
By NICHOLAS D. KRISTOF
¶ The National Catholic Reporter newspaper put it best: “Just days
before Christians celebrated Christmas, Jesus got evicted.”
¶ Yet the person giving Jesus the heave-ho in this case was not a
Bethlehem innkeeper. Nor was it an overzealous mayor angering conservatives
by pulling down Christmas decorations. Rather, it was a prominent bishop,
Thomas Olmsted, stripping St. ... 阅读全帖
f********g
发帖数: 6
41
来自主题: Biology版 - Re: 问个anneal linker的小问题
You don't need kinase if the vector/fragment the linker is ligated to has 5'p
on its end, which usually is the case. When you use restriction enzyme to
generate the new end from the linker, you also generate the 5'p on the end. I
think people who are using kinase are ligating oligos to de-phosphorylated
vector etc, in that case you need to add 5'p to your annealed oligos.

我以前anneal以后直接用来做连接,从来没有问题。而且anneal过程中我没有加过kinase
t****t
发帖数: 610
42
来自主题: Biology版 - 克隆小贴示。
CIP can solve the problem.
Case 1: no cut with either enzyme. The supercoil can be seperated by gel if
you run it long and slow enough.
Case 2: single cut. Although it is almost identical to the double cut
plasmid, it cannot be ligated to the insert, which has two different sticky
ends. The only possibility is self ligation. CIP can prevent or make it less
possible.
However, if you use blunt ends, that's another story and I agree with OP.
y******y
发帖数: 107
43
来自主题: Biology版 - 关于质粒构建问题
目的基因4.8kb, plasmid A 4.7kb。 ligation是用的pGEM-T easy vector的用来测序
的ligation方法,具体是
目的基因 150ng
plasmid A 50ng
T4 DNA ligase 1ul
2xbuffer 5ul
Total volume 10ul,
4C for 16h, 取2ul transformation。
s*******y
发帖数: 2366
44
来自主题: Biology版 - 跪求帮助:为什么克隆不出来
我也觉得没关系
insert大小对ligation影响不大
摩尔比关系也不大。。。
我觉得应该是酶切不彻底

跟你insert多长P关系也没有。。。
唯一的关系就是以后算ligation 摩尔比的时候可以考虑一下
s*******y
发帖数: 2366
45
来自主题: Biology版 - 跪求帮助:为什么克隆不出来
那就一个一个切
看看链的起来不
你ligate的时候没做self ligate的control吗?

惭愧。我是postdoc,实验室里就我一个人做分生。
双酶切, SacI和SalI,没有CIP处理。
这种情况下3:1链接过夜没问题吧?
只是想问问版上是否自己的设计本身就不合理。
主要是另外7个小的都出来了,这三个耽搁太久了。有些急。
O******e
发帖数: 14
46
来自主题: Biology版 - 克隆总有大片段丢失,请支招
两种都用过。大片段延长Heat Shock时间不知啥原理,有人有类似经验吗?
还有个实际问题就是,ligation反应之后放-20度,多久之后仍然可以用来做转化?感
觉如果ligate了,应该很稳定吧。大多数manual说可以overnight,有高手放了几天或
更久之后仍然成功转化的吗?我没试过,纯经验请教。
谢谢。
b******k
发帖数: 1874
47
so, if i am going to ligate two tandem short fragments (say, 50bp each):
synthesize 50nt oligo of Sense and antisense (with 5' phosphate group modifi
cation),
anneal them by boiling and cooling naturally,
then ligate the annealed fragments (ideally, with synthesizd 3' overhang fro
m first one, and 5' overhang from 2nd)by conventional T4 ligase system?
thanks.
R****n
发帖数: 708
48
来自主题: Biology版 - SNP Genotyping
多少个?几十还是几百?
几十的话,HRM, taqman.如果是genomic DNA可以考虑一下MPLA (multiplex probe
ligation amplification)在sequencer上作,一根管子能做三四十个位点
几百的话,single base extension, Ligation or Hybridization microarray. 公司
应该会帮你设计probe的.
C*******e
发帖数: 4348
49
2 个碱基保护未必够
我记得有个表格是说常用限制性内切酶分别需要几个碱基保护、酶切效率是多少
表格不在手边
不过你可以网上搜一下
另外也有可能像我上次遇到的,引物5'端合成的时候就少了
但是看你的描述我觉得更有可能的就是ligation不work
因为某些原因平板张长的都是false positive
ligation之后长满满的一般真不是好事
H****s
发帖数: 301
50
来自主题: Biology版 - 原核表达蛋白的毒性问题
I have tried DH5a/Top10/XL-1blue and it did not work neither.
I am very sure that there is no problem with my cut vector because I used it
to do library of huge size and never got any problem. I routinely use this
vector and same restriction sites for library cloning and never failed.
To make sure it is not cloning problem, I also did another control ligation.
Using the same materials, I ligated GFP fragment into the vector and got
tons of green colonies after induction. From the GFP control exp... 阅读全帖
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