C*******e
发帖数: 4348 | 1 谢谢这么详尽的trouble shooting
1. 限制性内切酶切E.coli plasmid显示，根据我们手上有的序列信息，如果只切E.
coli源的那
一半，酶切图谱就是正确的；如果在细菌X的origin那一段序列也切，酶切图谱就不能
完全吻合。所
以现在已经放弃用那个E.coli plasmid。
2.这个E.coli plasmid，同一管样品，别的人做PCR，还有ligation，酶切等等（都在E
.coli
源的那一半）是完全没有问题的，所以排除E.coli plasmid不够纯的问题。
3.同样的PCR master mix，用不同模板、引物，是可以PCR没有问题的，所以和试剂没
有关系。
4.引物序列等等已经确认过很多次；不过我没有确认到底有多少DNA在里面，谢谢提醒
，回头会查查
看。
5.要扩增的序列GC%是60%，不算特别高。PCR enhancer等等也试过，没有帮助。
6.我还提到如果不加DNA聚合酶的话加样孔里就没有东西；后来试过酚氯仿提取、乙醇
沉淀，再跑胶
只有一点smear，所以现在确定那是蛋白+核酸在孔里；为什么同样的master mix做别的
P |
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a****o
发帖数: 1786 | 2 dePi ur oligo
order a 5'P oligo A w/ known sequence
ligate w/ RNA ligase
use reverse complement oligo of A to do primer extension to get double
strand DNA
clone into T-vector or blunt end vector |
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C*******e
发帖数: 4348 | 3 我觉得省钱挺好的
不能因为钱不是你的就乱花
我们实验室现在的花钱习惯我就有点不适应
除了我都没有人自己配SDS-PAGE
都是买
我只有需要Gradient胶或者结果比较重要的时候才用买的
平时自己配一下没什么麻烦的；
他们每次表达蛋白的时候重新转质粒都用invitrogen买来的感受态细胞
一个反应一管
太tmd浪费了
要我说感受态细胞完全可以自己做
不放心的话ligation的时候用买来的好了；
连LB plate都没有人自己倒
全用买的；
一个LB还要买现成混好的粉末，
价钱贵了快一倍，
实验室明明几样原料都有。。。。。。 |
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g*****y
发帖数: 6325 | 4 为什么要这么高的浓度转化呢？ 我每次就切1ug. 胶回收，然后ligation, 转化都能挑
到正确得质粒。还有酶切一般我都不会
切超过2ug／tube. qiagen胶回收。 |
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s******y
发帖数: 28562 | 5 I think the empty plasmid from your negative control cells are actually due
to self-ligation.
You may need to treat your cutted plasmid with Alkaline Phosphatase for 5~10
minutes right after digestion and then recover the cutted produt on agarose
. |
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g*****y
发帖数: 6325 | 6 如果用2个酶来切，两个sticky ends是不同得，自己ligate得几率很低。 根本不需要
用phosphatase.
due
10
agarose |
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m*********7
发帖数: 5207 | 7 In principle, two different sticky ends should not self ligate.
in reality, it happens all the time!
I still don't understand why after so many years, and I keep using
phosphatase under that circumstances. |
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C*******e
发帖数: 4348 | 8 RNA ligase
RNA ligase可以催化RNA-RNA，RNA-DNA的ligation
而且其催化效率比DNA ligase高多了
可以说DNA ligase真是一个很烂的酶啊 |
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R****n
发帖数: 708 | 9 恩，老板搞了个NCI funded公司，做癌症早期检测，作临床样品。主要是建立个高灵敏
度的mutation/methylation screening.以前我们实验室有人做过array,Single base
extension和ligation 也就10%的灵敏度。 |
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c****l
发帖数: 1086 | 10 Are you talking about 5' RACE to let the product ligate into a loop? then
you will be able to design anther primer using known sequence. However in
the case of a transgene, it is no as simple as 5'RACE since very often the
transgene construct forms concatemers which greatly increase the complexity
of real products. there are several of these similar techniques such as "Pan
PCR", "selective circularization" or sth etc.
There are also commercial kits available.
Cre
Cre |
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p**********t
发帖数: 2636 | 11 克隆少是你转细胞的时候出了问题，篮白比例是ligation的问题 |
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s******y
发帖数: 28562 | 12 well, in order to change the AA in any position into another AA, you need to
cut open the peptide chain, take out one AA, then slip in another AA, and
then ligate the whole thing together. It require too much energy and not
really doable.
If the AA transferase can make a long peptide chain even just form a short peptide, then it is a breaking of the central dogma. However, all the transferase identified so far, can not make long peptide chain. |
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R****n
发帖数: 708 | 13 syber green原理是用allele specific PCR吧，一个primer 3'端用来区分phenotype.
我没做过不太清楚效果。还有什么和ligation结合的，HRM的。你说的太笼统了。
Taqman是用probe,两边一个dye一个quencher，不同的sequence dye不一样，一管PCR搞
定。这个基本上是业界的标准方法 |
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m******5
发帖数: 1383 | 14 Thank you ! I just entered a new lab, and I am doing a double sticky end
ligation which should be very easy before. I just have no idea of what is
going in my system. May be I over thought in some aspect…… |
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o********r
发帖数: 775 | 15 十年前俺做过这事（17Kb的plasmid），而且过程和你说的很象，最后筛了5个月（每天
就是早上抽质粒，下午PCR/跑胶，然后ligation/transformation，回家前pick colony
/incubation O/N），前后尝试了无数种ligase/competent cell，几乎市面上相关产品
都试了个遍，终于找到一个要的clone。其实17Kb对于某些质粒而言已经差不多是上限
，不稳定，体内很容易发生重组，如果可能尝试不同的vector，或者不同的competent
cell/transformation protocol。另外你或许可以试试Ca++ based
transformation。 |
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s**x
发帖数: 138 | 16 i did ligation and transformation. many colonies were seen on dish. then i
picked up colonies to grow. nothing grows. i tried 3 times and the last time
i used freshly prepared LB medium. any1 teaches me what is going on? what i
should do next?? thx |
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T**********t
发帖数: 1604 | 17 How did you choose your colony to pick?
How much volume of LB did you inoculate with a single colony and what type
and concentration of antibiotics are you using?
How long did you grow your LB culture? What temperature and speed?
Some general thoughts without any details regarding the above questions.
1）pour fresh LB plates, make fresh LB media
2) repeat ligation and transformation, make sure to include positive and
negative control
3) check plates to make sure you have healthy colonies and your... 阅读全帖 |
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v***a
发帖数: 1242 | 18 不知道呀。怎么样可以知道ligation等是否成功呢？我RE cut了以后跑胶然后切胶再提
取DNA再做transformation的。ligase几个月前用都是好的，现在应该也没问题吧？ |
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m**z
发帖数: 787 | 19 if your transformation is fine, means your competent cell is still competent
, I would probably just start from the beginning... that's what I usually do
if one round of cloning failed...especially when both vector and insertion
are newly cut and haven't been used for ligation before. |
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n********k
发帖数: 2818 | 20 simply modify the vector first by fusion PCR (about anywhere 200bp-1kb)
introducing the desired cut site at
the desired position...and then do whatever you want...I would't trust any
PCR with a 13KB template without
sequencing...if u are luck, everything might be alright but u could end up
in dark having no clue what's gone
wrong...If you want to do the cloning in a single step which I won't
recommend if not an experienced one, just
go three piece or even four piece ligation... that way, u mig... 阅读全帖 |
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f*******e
发帖数: 354 | 21 if he can insert MCS easily, he also can put his gene or some element into t
he single cut backbone.
i think the pcr protocol for such a big plasmid is not a good idea. why not
try enough enzyme digestion and dephosphate the backbone, in principle, 50%
ligation would be correct.
multiple |
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t********7
发帖数: 22 | 22 各位大侠，
我需要从一个bacteria的chromosome里克隆一段60kb的dna,并放到E.Coli里去表达。请
问有什么好的PCR方法能够克隆60kb的dna的。我目前只看到Roche好像有卖克隆5－20kb
的kit. 不知道有没有直接可以一次pcr 60kb的方法。（如果不行，是不是需要多次克
隆片段，然后ligation？）
还有，如果要放到e.coli表达，一般是用BAC做vector么？大家有相关什么文章可以推
荐阅读的嘛？或者BAC有哪里可以买的么？
先谢谢了。 |
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m******5
发帖数: 1383 | 23 I am doing a large fragment(2.3) plus large vector(12k) ligation.
I have learned previously that some plasmids containing certain sequence may
not be stable in DH5a line, but I didn't find online about which kind of
sequence or plasmid would be unstable.
Does anyone here have experience on handling this problem? |
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m******5
发帖数: 1383 | 24 同问！！！！！
特别是那套快速ligation kit里就有 |
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m**z
发帖数: 787 | 25 我总觉得ligation plate上面长得太满不见得是好事。。。 |
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v***a
发帖数: 1242 | 26 谢谢！那个表格我查到了。有一个问题啊，每个酶具体用什么保护碱基有章可循吗？比
如单是数量满足就行，还是碱基数量和种类要一样？
你说的ligation的问题我也觉得，那可能根子还在RE上。我再试试。多谢多谢！ |
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k*****o
发帖数: 1486 | 30 温度。DMSO。另外看不懂：“昨天colony PCR出了带结果质粒酶切后nothing” |
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s******n
发帖数: 47 | 32 看到一个protocol， 不太理解这句话
Illumina adapters with T-overhangs and customized to include 3-nt “barcodes
” were ligated to the cDNA？
请问Illumina adapters with T-overhangs and customized to include 3-nt “
barcodes” 是不是就是可以直接order的barcoded adapter？？
谢谢！！ |
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f**********e
发帖数: 1994 | 33 机器的 margin 太低,利润基本上大多是 reagents 来的。你无法想象
ILMN 的简单 library prep 是多大的竞争优势... 一个不稳定的 workflow
可以轻易毁掉一个几万块的 run, 还赔上几星期的时间。
PacBio RS 目前还没看到有任何人的使用报告。可能都是暗干在心里？ :)
Complete Genomics 的 library prep 会是他们的软肋，还有，他们会有
所有 SOLiD 有的问题, 因为他们也用 oligo ligation.
reagents |
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e****s
发帖数: 1125 | 34 感觉还是不能排除是克隆的问题。
载体不同，Ligation效率会差别很大。
要是没试过的话，可以试试看从那个真核载体里切出来，再克隆到原核载体里去。比
PCR片段直接克隆容易些。
你说的毒性问题，我个人认为可能性很小。可能我做的比较少，还没遇见过这样的情况。
DE3
（2 |
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m**z
发帖数: 787 | 35 you vector is clean cut, but what about your insert? In these strains, there
shouldn't be any leaky expression...
it
this
ligation. |
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C*******e
发帖数: 4348 | 36 敬仰
我看到ligation的那一段的时候终于看不下去了～～～～ |
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T**********t
发帖数: 1604 | 37 巨长是多长？太长的话你可以买一段短的RNA然后跟你的长的RNA片段ligate起来。 |
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w******e
发帖数: 1187 | 38 80base。
嗯，我把splint ligation的东西都买了，就是懒得做。。。 |
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s*******s
发帖数: 623 | 39 就是不用ligation
很多新方法出来用的实验室少，不是老板守旧就是人懒不愿意去试而已，其实有的方法
真的很不错 |
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c****9
发帖数: 95 | 40 我昨天刚收到的DNA序列测试结果，用软件分析后发现里面没有酶切的迹象。但是我之
前已经用过ECOR1酶切了,并且进行LIGATION。所以序列中应当有ECO SITE的。请问啥原因会导致这种情况的产生呢？谢
谢。 |
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n********k
发帖数: 2818 | 41 BamH1和Hind3在8里面中间就隔了1个
u run into a difficult but rather common problem...how did u do your
digestion? Have your checked NEB
appendix about it---the correct one would be digest with HindIII first and
then digest with BamHI,
otherwise, your chance of success is very low...That said, I would always
try to avoid this situation...go with
different enzyme cute sites, fusion PCR, homologue based cloning, or blunt
ligation(nowaday very
easy)...For a beginner, I hope to remind one truth: virtually for ... 阅读全帖 |
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n********k
发帖数: 2818 | 42 仅有几
个克隆，送过去测序，要做的8个构建株里面只有一个对的，其他基本上都是在Bgl2那
一段的引物地方要么有突变要么就是缺失。
well, could you start with these wrong ones now and just do some mutagenesis
, like you did with dpn1 ones...it would be much easier if it were due to
digestion/ligation etc...but it won't solve the problem if your fragments
are toxic or have too many repeats etc...It is a bit strange that it only
happen to your primer region in all the constructions though...if it is
toxic or due to recombination, it could happen to any regions or... 阅读全帖 |
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s********n
发帖数: 2939 | 43 不是很了解来龙去脉，你是没有带有不同tags的载体么？这样做克隆效率当然会很低啊。
如果你下一次做类似的课题，我建议你用Gateway cloning system，只需要将你的基因
克隆到pENTR vector（RE cloning，LIC cloning, TOPO cloning or BR
recombination），然后你就可以随意的将你的基因克隆到其他带有不同tag的pDEST
vectors上了，效率很高。
其他的还有ligation independent cloning (T4 DNAPol)和In-fusion cloning。 |
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s********n
发帖数: 2939 | 44 各有所长吧，我觉得QuikChange系列对于substitution和小片段的insertion和
deletion很有效，但对大片段的insertion和deletion效率很低；NEB的kitthroughput
要低一些（需要self-ligation），但对于大片段的insertion和deletion（尤其是
deletion）比较有效。 |
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n***w
发帖数: 2405 | 45 How do you do transformation?
using ligation product or the sequenced plasmid?
if you use sequenced plasmid and the concentration is around 200 ng/ul, you
simply can do a quick transformation (1ul DNA + 50ul competent cells (or
even 20ul), 20 minute on ice, 45 seconds 42 degree heat shock, 2 minute on
ice, then plate all of them).
and always set up controls.
可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（
25）或者更低（17）incubate 过夜？ |
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w******e
发帖数: 1187 | 46 Methods Mol Biol. 2009;504:385-98.
Using RNA aptamers and the proximity ligation assay for the detection of
cell surface antigens.
Pai SS, Ellington AD.
email: q************[email protected]
Thank you!! |
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p****s
发帖数: 3153 | 47 可以用native ligation，当然麻烦点，但是应该也不是没有，您说的那多半是ILV标记
法，但是那不利于assignment
当然了，大家不是一古脑的都做巨型蛋白，毕竟没有天然优势，不过去年有人用NMR研
究proteosome的机制我觉得还挺有
意思的，当然没有解结构了，我总是认为结构是重要的，但也不是唯一重要的问题。 |
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s***m
发帖数: 6197 | 48 用DNA做primer，合成RNA，遇到已有的RNA后有ligation的功能 |
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s***m
发帖数: 6197 | 49 用DNA做primer，合成RNA，遇到已有的RNA后有ligation的功能
之前发错图了，重发之，不好意思。 |
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s******y
发帖数: 28562 | 50 哦，不好意思一直没有看到你的问题。
我一向都用至少molecular ratio vector: insert < 1: 10
另外，如果可能的话，你的vector要用 alkaline phosphatase 处理一下，
然后insert最好在合成的时候要求他们加上磷酸集团，或者用 restriction enzyme 切开。
如果你做出来的克隆需要那么多的话（〉10 e7？是做random mutate library 吧？）
在我的经验中，需要至少10ug vector ( 大概含2ug ligated vector） |
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