M*****n
发帖数: 16729 | 1 molecular biology is not just pippetting.
an experienced molecular biology tech is so hard to find. |
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n***w
发帖数: 2405 | 2 谢谢。然后加完A后就可以直接做ligation了？还是说再纯化。。。？ |
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z*********8
发帖数: 1203 | 3 用过的达人请指点，我需要把3段DNA按照一定顺序克隆到plasmid上面，但是好用的
resctriciton enzyme在3段DNA里都有，所以ligation based cloning不太能用。然后
别人就介绍了这个infusion kit，发现克隆1个基因好用，克隆3个基因总是中间的那个
基因在，旁边的都不在，不知道有没有什么trick好克隆？谢谢大家！ |
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a********k
发帖数: 2273 | 4 花了一个小时，深深的鄙视一下自己的无聊行径！！
125 蔡亮 男 1980年11月 复旦大学 生命
科学 2007年12月毕业于[美国]北卡大学 [美国]加州大学旧金山分校 博士后
Cai L, Mostov K. Polarity is destiny. Cell. 2009 Nov 13;139(4):660-2. PubMed
PMID: 19914162; PubMed Central PMCID: PMC2900917.
Cai L, Makhov AM, Schafer DA, Bear JE. Coronin 1B antagonizes cortactin and
remodels Arp2/3-containing actin branches in lamellipodia. Cell. 2008 Sep
5;134(5):828-42. PubMed PMID: 18775315; PubMed Central PMCID: PMC2570342.
Cai L, Makhov AM, Bear JE. F-actin... 阅读全帖 |
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y******8
发帖数: 1764 | 5 才开张的新所吧。在北大，所是和学院平行的单位，据我所知，北大给新的所都会给一个5年一个亿的启
动支持。
夫妻店开的好才是好，学校也不用担心培养的人才什么时候为了家庭原因就飞走了。另
外，这个所的人才引进不错啊。这一位在千青中也是翘楚。
132 汪阳明 男 1979年12月 北京大学 生命
科学 2006年02月毕业于[美国]伊利诺伊大学厄巴纳－尚佩恩分校 [美国]加州大
学旧金山分校 博士后
1: Wang Y, Blelloch R. Cell cycle regulation by microRNAs in stem cells.
Results
Probl Cell Differ. 2011;53:459-72. PubMed PMID: 21630156.
2: Wang Y, Blelloch R. Cell cycle regulation by MicroRNAs in embryonic stem
cells. Cancer Res. 2009 May 15;69(10):4093-6. Epub 2009 May 12.... 阅读全帖 |
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s********r
发帖数: 312 | 6 Yes, I CIPed the vector for 1h, but it didnt appears to be self ligation.
The 3kb plasmid is weird, because the vector itself is 12kb. |
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a****d
发帖数: 1919 | 7 楼上的，大片段backbone用胶连接，能不能具体解释一下，和ligation有区别吗？ |
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l**********1
发帖数: 5204 | 8 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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C***Y
发帖数: 61 | 9 After linker (~120 bp) ligation, fragments of 200-450 bp will be cut from
gel for purification, so your sonication result is very good. |
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C***Y
发帖数: 61 | 10 After linker (~120 bp) ligation, fragments of 200-450 bp will be cut from
gel for purification, so your sonication result is very good. |
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l**********1
发帖数: 5204 | 11 LZ please try below (I) to (IV) four strands overlap PCR
your target 7kp=2kp(I)+2kp(II)+2kp(III)+1kp(IV)
if you did not know downstream any conservative sequence information
your 3' RACE can use ligation-anchored PCR
就是用内切酶
//en.wikipedia.org/wiki/Restriction_enzyme
对应的人工引物锚定long mRNA 的3'端 做人工锚定引物
i.e. by NotI it should be inserted to 3'端 5'GC---GGCCGC
from
//en.wikipedia.org/wiki/Restriction_enzyme
Enzyme Source Recognition Sequence Cut
NotI Nocardia otitidis
5'GCGGCCGC
3'CGCCGG... 阅读全帖 |
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l**********1
发帖数: 5204 | 12 Please go to
//www.ncbi.nlm.nih.gov/pmc/articles/PMC2518226/
>Overall, our results suggest that PUB22 and PUB23 coordinately control a
drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.
Subcellular Localization
The sGFP cDNA clone was fused in-frame to the 59 end of the full-length
PUB22 and PUB23 coding region and ligated into the pBI221 transient
expression vector. The sGFP-PUB22 and sGFP-PUB23 fusion constructs
were transformed into protoplasts prepared from wild-typ... 阅读全帖 |
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l**********1
发帖数: 5204 | 13 if your target mRNA its 3' UTR super long (over several kp) tor before the stop codon high GC rich region
then you can only get with normal qRTPCR then PCR whole 3RACE PCR products.
This case please try another method:
: ligation-anchored PCR cloning
一句话就是用内切酶对应的人工引物锚定: 3'端 做人工锚定引物PCR
mRNA with a primer (3'-poly
(dT)17 with an anchor sequence at its 5'end, then qRTPCR then PCR sub cloning... sequencing.
References:
//genome.cshlp.org/content/4/1/19.full.pdf
//www.ncbi.nlm.nih.gov/pmc/articles... 阅读全帖 |
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l**********1
发帖数: 5204 | 14 楼主 没有确定的可以general qRTPCR的两组碱基序列才会问这个贴的吧
有时候就得用特殊的anchor adaptor ligation qRTPCR and relative PCRs
details steps 请看图
snap copied and pasted from
//www.ncbi.nlm.nih.gov/pubmed/14660366
NB: for which kind of restriction enzyme that fit to your target,
case by case just by your PCR cycling and Gel Image and sequencing cycles again and again
and your fate probability. |
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y*****w
发帖数: 146 | 15 需要克隆几个genes 到 pMXs-IRES-Blasticidin vector, 试了几次都没有得到克隆子
，已经验证了应该不是ligation的问题。想问问有没有人用过pMXs-IRES-Blasticidin
vector，是不是有什么tricks在里面。十分感谢！ |
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s******s
发帖数: 13035 | 16 关于3，你是技术更新了老的方法都忘了。在next gene之前，用测序测表达，
就把cDNA加接头，然后切成固定大小片段，然后几十个ligate在一起，作为
一个反应去测序。nanopore显然可以用这样的方法，也就是样品要处理一下
而已，这个比library还是简单
板？ |
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b******r
发帖数: 111 | 17 Hi,everyone,
I have to use single restriction sites for insertion of multiple fragments
. When using KpnI site, just get one successful insertion from 70 clones.
For NotI, it is 1 from 4,BamHI is 1 from 2. My question is why the ligation
is so bad at KpnI site? Or other reasons? Thanks |
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b******s
发帖数: 1089 | 18 实验室其他人遇到过经年的KpnI不work。但我自己切没遇到过问题。单酶切你可以上很
浓的质粒，多加点酶，但是不要超过反应体系的10%，过夜，加CIP，应该能做出来。
fragments
ligation |
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l**********1
发帖数: 5204 | 19 linker-based PCR
plus
iMapper soft:
//www.ncbi.nlm.nih.gov/pubmed/18974167
//geocachingsoftware.com/imapper.html
more details
please to to E-Book
Chapter 2:
its link:
FTP://ftp.sanger.ac.uk/pub4/theses/kong/chapter2.pdf
citation:
>Based on these expectations, a web-based server called iMapper (Insertional
Mutagenesis Mapping and Analysis Tool) was developed for the efficient
analysis of insertion site sequence reads against vertebrate and
invertebrate Ensembl genomes. Taking linker-based PCR se... 阅读全帖 |
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k****l
发帖数: 279 | 20 Site-directed mutagenesis
or cut that piece out, PCR that piece from a right plasmid,do a ligation |
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N2
发帖数: 81 | 21 1)screen 3-5 more colonies and verify the sequence of that site by
sequencing.
2)if there is no correct one, it seems the fragment used for ligation having
that mutation.
3) do a reverse PCR to fix it if you plasmid is not too big.
4)site mutegensis by Quick Change
5)rebuild the plasmid with new fragments from PCR using Phusion DNA
ploymerase
from 3) to 5) find any one which will be easy for you.
good luck! |
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b******s
发帖数: 1089 | 22 做cloning，我觉得要是有问题，干脆重做。要是只是插入片段上有突变，重新扩增，
酶切，ligation也不算麻烦。一时偷懒，下面的步骤可能麻烦会更多，经常到最后还要
重新来过。 |
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q**********0
发帖数: 335 | 23 Recently I tried three different TA cloning Kits from Invitrogen (TA cloning
, TOPO TA clong and dual promoter TA cloning). I found all of them have very
high self-ligation rate (>90%). I don't know anybody have similar results?
Any suggestions for other better choice for TA clone? Thanks. |
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e****s
发帖数: 1125 | 24 I see.
Apparently, your new constructs are just not right based on your digestion
tests, which explains the expression problem.
Digestion tests is always better than PCR to confirm the subcloning
constructs.
I only sequence samples after digestion tests.
Sometimes I struggle with the ligation of big constructs as well.
Good luck!
sent
).
new |
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r****r
发帖数: 36 | 25 Indeed, it's more difficult to manipulate big constructs than small
constructs with ligation method. So sometimes I used yeast recombination to
handle big constructs. The drawback of using yeast is the high mutation rate
. I always have to use several independent clones to confirm my observation
was really due to my mutations instead of random mutations caused by yeast. |
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R****n
发帖数: 708 | 26 METHYL-SEQ送去core facility一年还没有结果，最近一次F５和BARCODE都没有问题，F
３LIGATION ３个CYCLE就没有信号好了。NANODROP和跑胶都确认LIBRARY没有问题，也
没有PRIMER DIMER。用的是SOLID 5500系统。 |
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L*****t
发帖数: 56 | 27 连Pipette都不会用的小本反而好带，一张白纸，示范几次就能学的很像样了。这种半
桶水晃荡的最难。水浴锅的使用演示过了，也说过这种小体积，短时间的的反应最好用
水浴锅，控温效果比PCR机和Heating Block好，但是到实际操作就自说自话的变成25C=
室温，37C=大肠杆菌的培养箱。我估计她是觉得水浴锅加热太慢，懒得等而已。
还有很多小事说过了第二天肯定记不住，再讲再忘。比如样品我让她放在冻存盒里再放
-20C冰箱，但是经常是做完实验往4C冰箱某个角落里一塞就走了，第二天再到处找。菌
种不好好保管，平板不倒置结果弄得到处都是水，性格再好的人也要怒
酶全扔掉是只因为保存不当，而是她没有告诉任何人就悄悄地一个人开始做ligation，
很担心这几个酶是不是也被污染了。 |
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l**********1
发帖数: 5204 | 28 RE LZ:
pls try
Cassette-ligation downstream 3'RACE PCR
or Tail-PCR
Protocol:
看图说话
>
Amplification of FSTs by 3’-RACE
Total RNA was isolated using hot-phenol extraction
[27], from 20 ml cultures in 50 ml Falcon tubes on a rotating
wheel. Reverse transcription used the SuperScript
III First-Strand kit from Invitrogen in a DNA Engine
PCR machine (MJ research). Primer QT was mixed with
5 μg RNA, incubated at 65 °C for 5 min then cooled to
55 °C over 100 sec, after which the annealed mixture
was kept... 阅读全帖 |
|
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I*****y
发帖数: 6402 | 30 it's going to be blunt end anyway |
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s******y
发帖数: 28562 | 31 我倒。。。不行。因为你的PCR enzyme 会把序列都补平的。如果是说的是
TA cloning那另当别论。 |
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|
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A******y
发帖数: 2041 | 34 Nothing wrong or new with this. You either collaborate with people that can
do the experiment or figure out your own way. Some techniques are harder
to do such as ligation mediated PCR and chromatin reconstitution (which some
trick were kept in the original lab to make sure the competitor will take
longer time to figure out how to do it). Although, I'm reviewing one paper,
the guy literally told me his/her lab cannot do a Western blot and it will
cost 20k for someone to do it...I just about t... 阅读全帖 |
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V**3
发帖数: 12756 | 35 哈哈哈哈
你还扯上啥纳税人的钱了，笑死人了
美国纳税人的钱被糟蹋的成吨成吨，你一个千老敢去指责谁了？ 就看一个新生觉着你
能压在他头上一点，赶紧用用这种优越感
说白了你就是当年自己倒霉过来现在还在倒霉着，所以看不得别人过的比你舒坦而已
你这种人万一熬上教授，哈哈，就直接排行榜上见了 |
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V**3
发帖数: 12756 | 37 别人给他指出的他根本不会理性考虑的
一个千老被老板指派带学生，学生爱咋咋，跟你有啥关系啊。 别人说奖学金也不是你
出的，他还扯上纳税人钱了，真可笑
楼道里扫楼的老黑偷懒耍滑你让他去指责指责看看 |
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d**p
发帖数: 149 | 38 Can you find the restriction enzyme site less than 2kb distance from the
insertion site? If yes, you do not have amplify very long fragments. Similar
size of fragments can joint together easier than different size fragments.
Another way: You can amplify the whole plasmid( How big is the plasmid?) by
the primers at the insertion site. Then you can introduce the enzyme site in
the primer. After PCR amplify the plasmid, digest it and ligate your 800bp
fragment and transform. You need to amplify you... 阅读全帖 |
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a********k
发帖数: 2273 | 39 分两段PCR，做3-way ligation。
8482 |
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c*********r
发帖数: 1312 | 40 谢谢，我之前有点担心3-way ligation效率低，想一步搞定。有推荐的protocol吗？ |
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o**a
发帖数: 86 | 42 【 以下文字转载自 Chemistry 讨论区 】
发信人: ouba (comfortably numb), 信区: Chemistry
标 题: how to block dna 3' OH
发信站: BBS 未名空间站 (Mon Apr 22 16:45:47 2013, 美东)
Anyone knows of a simple reaction to block the dna 3'OH from ligation to a
free 5'ppp? Thanks! |
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X***n
发帖数: 366 | 43 No way. If you don't mind add another nt to the 3'-end of DNA, you can
ligate a ddT to the 3'-end fist. |
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l*******e
发帖数: 170 | 44 1. 我现在要克隆一个基因X的promoter序列，软件分析了一下，大约1500kb的样子
2. 买个kit, genome DNA isolation（谁给推荐一个？）
3. 纯化genome DNA
4. 设计引物PCR这个大约1500bp的片段，在TSS前面
5. 然后ligate到一个luciferase的reporter vector里
没做过这玩意，大家帮忙看看哪里不对 |
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l*******e
发帖数: 170 | 45 1. 我现在要克隆一个基因X的promoter序列，软件分析了一下，大约1500kb的样子
2. 买个kit, genome DNA isolation（谁给推荐一个？）
3. 纯化genome DNA
4. 设计引物PCR这个大约1500bp的片段，在TSS前面
5. 然后ligate到一个luciferase的reporter vector里
没做过这玩意，大家帮忙看看哪里不对 |
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W*V
发帖数: 13 | 46 我周一作了四个TRIP-LIGATION 克隆, 筛选都阳性. 今天又作了六个. 集我30年的分
子生物学的工作经验, 我有一套简易,有效的基因克隆方法. 无论片断大小 (>8Kb),
无论酶切类型, 无论何种载体 和感受态细胞, 全都工作,没有问题. 可惜因为版权
关系 (正在写书),其操作步骤现只能非免费提供. 该克隆方法(章节) 转让费为$100.
为证明其可信度,我可以为你作一免费克隆. 你只提供目标片断, 我在三天内交付你
克隆好的质粒. 有兴趣的话,可联系,用此邮箱. |
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W*V
发帖数: 13 | 47 我周一作了四个TRIP-LIGATION 克隆, 筛选都阳性. 今天又作了六个. 集30年的分子生
物学的工作经验, 我有一套简易,有效的基因克隆方法. 无论片断大小 (>8Kb), 无论酶
切类型, 无论何种载体 和感受态细胞, 全都工作,没有问题. 可惜因为版权关系 (正在
写书),其操作步骤现只能非免费提供. 该克隆方法(章节) 转让费为$100.为证明其可信
度,我可以为你作一免费克隆. 你只提供目标片断, 我在三天内交付你克隆好的质粒.
有兴趣的话,可联系,用此邮箱. l******[email protected] |
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m****M
发帖数: 360 | 48 Can you describe it in detail? If 5' is missed, how did you clone the gene (
T-vector ligation)? If 15nt is missed at 3' how the primer complement with
your DNA plate correctly? If it complemented at right position how did you
know 15nt is missed sine it will be identical to one of your DNA strands?
tried
similar
news |
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N****n
发帖数: 294 | 49 A PCR was performed and cloned directly into vecotr by ligation (primer
phosphorylated first). Sequencing indicated that one primer was missing 15nt
from the 5' end. Because the 3' end of the primer is intact, that is why
the PCR worked fine.
( |
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q***7
发帖数: 144 | 50 Use this company's gel extraction kit will allow you get washing buffer-free
linearized vectors/DNA fragments, which will increase your cloning
efficiency.
You can store linearized DNA for over 5 years at -20C and still can ligate
to corresponding DNA fragment for over 90%.
http://www.greenbioresearch.com/gel-extraction-pcr-purification |
|
