s******a
发帖数: 472 | 1 又来请教大家关于recombineering的问题了。因为重组是一个小概率事件（就算在重组
酶存在的情况下），所以就需要有筛选的策略。最简便高效的方法可以导入一个loxP-
Neo-loxP用于positive selection，最后可以利用cre重组酶去掉Neo。缺点是会留下一
个loxP位点，也不利于在该位点进行进一步的遗传操作。
鉴于此，我们决定采用positive/negative selection的方法进行recombineering。2种
常用方法包括galK筛选和rpsL-Kana筛选。我目前采用的是rpsL-Kana方法，Kana 使细
菌对Kanamycin有抗性，rpsL是细菌对streptomycin敏感。第一步recombineering
select “kana resistance”；第二步reombineering counter-select rpsL。
经过第一步，克隆PCR鉴定可以有95%正确率，即基本上所有克隆都带有重组的rpsL-
Kana。但是第二步（将rpsL-Kana替换为insert），获得阳性克隆如同中彩票一样，概
率很低。筛200... 阅读全帖 |
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n********k
发帖数: 2818 | 2 it is a totally different question now...u are talking about two
recombination events or hope to have one non-
perfect recombination event so that u can get in both ur knockin arm and
another 100bp with ur long arm...I
don't know, it could be tricky..I have a friend who did similar things but
used double selections... don't really
have experience with this, so maybe someone else could help...BTW, u could
have asked question more
clearly...no biggie |
|
n********k
发帖数: 2818 | 3 Finally u came:)))...I also have a question in this case, so will the
efficiency of desired targeting much lower, or
not really with your experience... Another question is what if the middle
fragment is like 10kb or even more....I
was thinking it would take two recombination events to get them in...then
the efficiency would be really low...
recombination
may
maximize
floxed |
|
z*t
发帖数: 863 | 4 有人说3kb以上的insert从entry vector到destination vector recombination rate会
很低，求问3kb以上的insert recombination后postive rate如何？谢谢：） |
|
h***9
发帖数: 662 | 5 发生recombination的时候，除了两个loxp之间的sequence会被recombine掉，loxp外端的序列也可能会受影响么？我在设计一个knock in mouse 的targeting vector,打算把loxp放在intron里面，但不知道loxp两边是否应该加一些random sequence.谢谢。 |
|
l******o
发帖数: 103 | 6 I have the following problem. I want to introduce a point mutation to my
plasmid. The plasmid is super big. So I first cut out a fragment(~4kb) with
XbaI and then ligate it to another vector. After introducing mutation with
mutagenesis kit, the sequencing result is as expected.
Next step is to put the XbaI-XbaI fragment containing the mutation back
into the original clone. After ligation and transformation with DH5alpha I
get a number of colonies (~20) but no colonies in the negative control. B... 阅读全帖 |
|
f*****y
发帖数: 464 | 7 两个概念最主要的差别在哪里？碰到一个非分子生物专业的考试题如下：
A geneticist has artificially produced a microorganism that contains a new
combination of genes. He wants to know if recombination or transposition has
occurred. The major difference between these two processes is that
recombination involves which of the following?
A) DNA repair
B) Endonuclease sites
C) Gene rearrangement
D) Homologous sites
E) Sites on the same chromosome
多谢！ |
|
|
|
G*******1
发帖数: 137 | 10 Human βhsp90 as adjuvant in HCV/ HBV Recombinant vaccine
需要病毒免疫/疫苗相关专业的，感兴趣的请发简历到z******[email protected] 。非相关
专业的请勿扰。谢谢。 |
|
j********o
发帖数: 2 | 11 BL21codon+ is indeed BL21 with a plasmid that expresses rare codons and
BL21(DE3)pLysS/E is indeed BL21 with a plasmid that expresses an enzyme that
degrade T7 polymerase to avoid leaky. Both plasmid are chlorepenicol
resistance. In inder to make codon plus in BL21(DE3)pLysS/E strain, drug
marker of one plasmid must be change and the system will become too
complicated - the strain resist to two drug, plus another drug marker for your
recombinant protein.
it.
are
is
no
of |
|
j*****q
发帖数: 82 | 12 大大们进来看看
两条homo arm一定要一模一样的序列吗？
如果其中一条（长的那条，大概6kbp）中间有大概100bp不到的不同序列，会影响
recombination的efficiency吗？
多谢 |
|
H****N
发帖数: 997 | 13 It should work. Just be aware that you will have two possible recombination
events: in one event, the middle fragment is used and therefore you
insertion will not be introduced; in another, the fragment on the side is
used and you will have the desired allele with you insertion. You have
to have a genotyping strategy that can distinguish these two events. You may
also increase the length of the homologous fragment on the side to maximize
your chance of success. This is actually very similary to |
|
H****N
发帖数: 997 | 14 Hey how is it going? To your first question, the efficiency will be lower,
but not that much lower. Say if you have 3 kb on the side and 2kb in the
middle, you may have half the efficiency of what you would without the
insertion. As to the second question, if you have a 10kb fragment in the
middle, the chance of getting the desired recombination may be very low. It'
s just my gut feeling and I don't have any actual experience with a long
middle arm. If I were you, I would give it a try, if the |
|
A******y
发帖数: 2041 | 15 Recombinant protein is around 300 a pop...if you need a lot of protein, you
need to make it yourself. Also, does activity matter? If you are doing
structure, making protein should be a piece of cake or you are in the wrong
lab. Or you can call the company for a larger scale quote. I used BPS biosciences, and the owner will do his best to make a sell and will try to make thing works, but I bet it will still cost you.
or try addgene and hope the plasmid of interest is deposited and make it
you |
|
b******r
发帖数: 111 | 16 I am cloning 3.4kb human genome DNA into a 8.4kb vector.Isolate some
plasmids. Recut,then 3.4kb bands
appear. But sequencing shows it is not 3.4kb human DNA,it is a unknown
fragment. Strangely,the size is same
as 3.4kb! What happen? Any idea to eliminate recombination ? The competent
cells used are Invitrogen ElectroMAX™ DH10B™ T1 Phage-
Resistant Competent Cells Cat. No. 12033-015. |
|
M*****n
发帖数: 16729 | 17 if you are concerned about recombination, use Stbl4 cells. |
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m******s
发帖数: 1 | 18 我们想找个公司帮我们提human recombinant protein,最好是untagged，并且是从
human cell line里面提纯的，大家有没有什么公司可以推荐？谢谢！ |
|
b******s
发帖数: 1089 | 19 如果只是单个entry vector到destination vector，效率很高。
我做过大于6KB的都没问题。
但是我做multisite 3-way recombination效率极其低，还有很多false positive。 |
|
h**********r
发帖数: 671 | 20 Title:
Functional characterization of eight human cytochrome P450 1A2 gene variants
by recombinant protein expression
The Pharmacogenomics Journal 10, 478-488 (December 2010) | doi:10.1038/tpj.
2010.2
My email is m******[email protected]. Thank you so much! |
|
n***3
发帖数: 663 | 21 可能这里知道的人不多，想问一下用Kluyveromyces lactis （一种yeast，在工业上用
）生产recombinant protein的大型工业生产的方法哪里能找到啊？
thanks |
|
s******a
发帖数: 472 | 22 我用的是SW102, 估计不同genetic background的strain对streptomycin的敏感性也不
同。 早先报道rpsL-kana筛选的paper他们用1mg/ml；但是genebridge公司
recombineering kit用rpsL-neo，他们用50ug/ml。怎么有这么大的差别呢。
我后来测了一下OD，2.5mg/ml streptomycin只能使OD600降低20%左右。
streptomycin不能完全抑制细菌生长，但是到底多大溶度才合适进行counter-
selection呢？ |
|
m******p
发帖数: 67 | 23 Need to make a recombinant protein. If the signal peptide should be included
if to express it in E coli or in mammalian cells?
thanks. |
|
e****s
发帖数: 1125 | 24 I would check plasmid and expression more carefully before draw a conclusion
of genomic recombination.
1. Have you check the whole 4kb cDNA after site-direct mutagenesis? It will
take you 5 sequencing to check the whole cDNA.
2. How do you check the expression? Have you check both supernatant and
total lysis? by western or just SDS-PAGE. The point is single mutant could
dramatically decrease the expression.
3. Purify plasmid from the expression E.coli, do cutting test and send for
sequencing.
w... 阅读全帖 |
|
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. |
|
f*****y
发帖数: 464 | 26 为什么呢？non-homologous recombination不也存在吗？ |
|
j****n
发帖数: 3370 | 27 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 |
|
l****6
发帖数: 6 | 28 Is there a study on meiosis recombination frequency in the genome level?
We have a model to address the question, but we are not in the field, is it
an important biology question?
Thanks for inputs! |
|
l****6
发帖数: 6 | 29 Thanks for your response.
My question: how important if I can show in genome level, the meiosis
recombination frequency? Have similar work been done? |
|
n*****n
发帖数: 86 | 30 Heard this from a Stanford guy during a seminar, about 200 recombination
events per generation, but I am still not sure if it's on the sperm side or
the egg side
it |
|
n********k
发帖数: 2818 | 31 As you folks are discussing Cori, I happened to read his autobiography and
find it both interesting and enlightening:)))...BTW, it sounds Richard Axel
is a lot nicer than what I/we have heard...or in another word, his cruelty
is rooted in his obsession with science and indifference to humanity:))); As
well, it is pretty...he noted Cori but left his ex out...pretty human or
not human?
Autobiography
Richard AxelNew York City is my world. I was born in Brooklyn, the first
child of immigrant parents... 阅读全帖 |
|
C*I
发帖数: 4736 | 32 一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须
经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人
民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过
其它受体/宿体，而直接传染给人。 他们... 阅读全帖 |
|
C*I
发帖数: 4736 | 33 一直在误导，现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人，必须
经过一个中间宿体性的其它动物才能传染给人。所以，病毒发生后，就故意误导全国人
民去海鲜市场找证据，找其它野生动物的麻烦。 而且还是病毒所去找的，找完了还装
模做样化验呀，分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
，一直不敢说，说了其它相关已经在人就会去早那种动物监测。 所以压根不说，打马
虎眼。
事实上，早在2013 年，就是这个武汉病毒研究所，已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒，其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体，并能够感
染人的细胞。对此新发现，武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”，以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说，从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus， 可以不经过
其它受体/宿体，而直接传染给人。 他们... 阅读全帖 |
|
C*I
发帖数: 4736 | 34 Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the
ACE2 receptor
Xing-Yi Ge, Jia-Lu Li, Xing-Lou Yang, Aleksei A. Chmura, Guangjian Zhu,
Jonathan H. Epstein, Jonna K. Mazet, Ben Hu, Wei Zhang, Cheng Peng, Yu-Ji
Zhang, Chu-Ming Luo, Bing Tan, Ning Wang, Yan Zhu, Gary Crameri, Shu-Yi
Zhang, Lin-Fa Wang, Peter Daszak & Zheng-Li Shi
Nature volume 503, pages535–538(2013)Cite this article
Abstract
The 2002–3 pandemic caused by severe acute respirator... 阅读全帖 |
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S*****a
发帖数: 63 | 35 【 以下文字转载自 Pharmaceutical 讨论区 】
发信人: Somalia (Anti-Pirates), 信区: Pharmaceutical
标 题: Biologics Discovery and Development Positions Available at Abpro Labs in Woburn, MA
关键字: Biologics,Discovery,Development,Antibody,Computational
发信站: BBS 未名空间站 (Wed May 31 20:28:52 2017, 美东)
Multiple Biologics Discovery and Development Positions Available at Abpro
Labs in Woburn, MA
www.abpro-labs.com
To apply please send resume to [email protected]
1. Scientist/Senior Scientist, Antibody Display Technology
... 阅读全帖 |
|
S*****a
发帖数: 63 | 36 【 以下文字转载自 Pharmaceutical 讨论区 】
发信人: Somalia (Anti-Pirates), 信区: Pharmaceutical
标 题: Biologics Discovery and Development Positions Available at Abpro Labs in Woburn, MA
关键字: Biologics,Discovery,Development,Antibody,Computational
发信站: BBS 未名空间站 (Wed May 31 20:28:52 2017, 美东)
Multiple Biologics Discovery and Development Positions Available at Abpro
Labs in Woburn, MA
www.abpro-labs.com
To apply please send resume to [email protected]
1. Scientist/Senior Scientist, Antibody Display Technology
... 阅读全帖 |
|
S*****a
发帖数: 63 | 37 【 以下文字转载自 Pharmaceutical 讨论区 】
发信人: Somalia (Anti-Pirates), 信区: Pharmaceutical
标 题: Biologics Discovery and Development Positions Available at Abpro Labs in Woburn, MA
关键字: Biologics,Discovery,Development,Antibody,Computational
发信站: BBS 未名空间站 (Wed May 31 20:28:52 2017, 美东)
Multiple Biologics Discovery and Development Positions Available at Abpro
Labs in Woburn, MA
www.abpro-labs.com
To apply please send resume to [email protected]
1. Scientist/Senior Scientist, Antibody Display Technology
... 阅读全帖 |
|
p*****c
发帖数: 20445 | 38 Marc R. Freeman, Ph.D.
HHMI Early Career Scientist
University of Massachusetts Medical School
Neurons hog the attention, but our brains couldn’t operate without the
unassuming neural cells called glia. Marc Freeman is working to bring these
overlooked cells—which represent about 60 percent of brain cells—their
share of credit.
Freeman has been dissecting the role of glia in brain development, function,
and healing after injury, when glia hurry to the damaged area and devour
cellular debris to al... 阅读全帖 |
|
S*****a
发帖数: 63 | 39 【 以下文字转载自 Pharmaceutical 讨论区 】
发信人: Somalia (Anti-Pirates), 信区: Pharmaceutical
标 题: Biologics Discovery and Development Positions Available at Abpro Labs in Woburn, MA
关键字: Biologics,Discovery,Development,Antibody,Computational
发信站: BBS 未名空间站 (Wed May 31 20:28:52 2017, 美东)
Multiple Biologics Discovery and Development Positions Available at Abpro
Labs in Woburn, MA
www.abpro-labs.com
To apply please send resume to [email protected]
1. Scientist/Senior Scientist, Antibody Display Technology
... 阅读全帖 |
|
S*****a
发帖数: 63 | 40 Multiple Biologics Discovery and Development Positions Available at Abpro
Labs in Woburn, MA
www.abpro-labs.com
To apply please send resume to [email protected]
1. Scientist/Senior Scientist, Antibody Display Technology
We are seeking an experienced and highly motivated Scientist with strong
hands-on experience in antibody display (phage/yeast) to support discovery
research projects by screening to identify lead candidate therapeutic
antibodies, as well as engineering molecules for improved... 阅读全帖 |
|
z*******4
发帖数: 285 | 41 算了，你脑子大概也被活摘过
懒得跟你解释，贴个清单吧：
累计研究数量 注册号 注册日期 研究者单位 研究者单位（英文） 注
册题目 注册题目（英文) 预计纳入参试者日期
1 ChiCTR2000029308 1/23/2020 武汉市金银潭医院（武汉市传染病医院）
Wuhan Jinyintan Hospital (Wuhan Infectious Diseases Hospital) 一项评价
洛匹那韦/利托那韦和干扰素-α2b联合治疗武汉新型冠状病毒肺炎(COVID-19)住院患者
的疗效和安全性随机、开放、空白对照的研究 A randomized, open-label, blank-
controlled trial for the efficacy and safety of lopinavir-ritonavir and
interferon-alpha 2b in hospitalization patients with novel coronavirus
pneumonia (COVID-1... 阅读全帖 |
|
l**********1
发帖数: 5204 | 42 RE LZ
pls refer
PMID 21063464
Liu Y et al.
Tamoxifen-independent recombination in the RIP-CreER mouse. (2010).
PLoS One. 25: e13533.
http://www.ncbi.nlm.nih.gov/pubmed/21063464
>There are at least two possible explanations for
>these discrepancies.
>One possibility is that after many generations
of backcrossing to the C57BL/6 background, the expression of the
randomly integrated RIP-CreER transgene is enhanced, thus
increasing the probability of its nuclear entry, even in tamoxifenuntreated
cel... 阅读全帖 |
|
w********o
发帖数: 10088 | 43 恩，假设都是在ideal diode那个区间测吧。偏离1大部分时候是由space charge region的recombination引起的，neutral region的recombination得到的ideality factor还是1. 我最近的测试好像如果一边是heavy doping，增加另一边 doping有点用，不过不知道是不是process的noise引起的
理论上说SR recombination rate=1/tau*（np-ni^2）/(n+p+2ni).如果p>>n，基本上可以近似成 1/tau*(n-ni^2/p)/(1+2ni).增加n，recombination rate升高。不过前提是tau不变。如果看整个recombination，貌似还应该考虑电子通过整个space charge region的时间，这么又复杂了。 |
|
发帖数: 1 | 44 xiaxianyue生物不行吧？dna重组只会使得遗传概率更加趋向于1/2的幂，而不是相反
假设精子的23个染色体里，不失去一般性，来自父亲的染色体个数是a < 23/2，来自母
亲的染色体个数是23-a > 23/2
假设所有染色体发生recombination的概率相同，那么在父系a个染色体发生
recombination的总量，应该小于母系23-a个染色体的recombination总量。
所以，父系dna总量因父系染色体recombination而减少的量，要小于因母系染色体
recomibation而增加的量，因此总共获得的父系dna数量比例，要大于a/23，因此会更
偏向于50%。 |
|
发帖数: 1 | 45 xiaxianyue生物不行吧？dna重组只会使得遗传概率更加趋向于1/2的幂，而不是相反
假设精子的23个染色体里，不失去一般性，来自父亲的染色体个数是a < 23/2，来自母
亲的染色体个数是23-a > 23/2
假设所有染色体发生recombination的概率相同，那么在父系a个染色体发生
recombination的总量，应该小于母系23-a个染色体的recombination总量。
所以，父系dna总量因父系染色体recombination而减少的量，要小于因母系染色体
recomibation而增加的量，因此总共获得的父系dna数量比例，要大于a/23，因此会更
偏向于50%。 |
|
发帖数: 1 | 46 英文媒体也报道了
https://apnews.com/Business%20Wire/fe24e251ddb843a9b091561a06f8eead
Clover Initiates Development of Recombinant Subunit-Trimer Vaccine for Wuhan
Coronavirus (2019-nCoV)
CHENGDU, China--(BUSINESS WIRE)--Jan 28, 2020--
Clover Biopharmaceuticals, a global clinical-stage biotechnology company
focused on developing novel and transformative biologic therapies, today
announced that it has initiated development of a recombinant subunit vaccine
for 2019-nCoV, the newly identified coronavirus tha... 阅读全帖 |
|
b****n
发帖数: 8 | 47 Address:
Email:
Cell phone:
Work phone:
Oct 28, 2013
Department of Homeland Security
USCIS Nebraska Service Center
P.O. Box 87103
Lincoln, NE 68501-7103
I-140 Immigration Visa Petition Under First Preference EB-1
Petitioner/Beneficiary: XX Z., Ph.D.
Category of Petition: Alien of Extraordinary Ability
Classification Sought: 203(b)(1)(A)
Nature of Submission: Original Submission
Dear Immigration Officer,
This letter is respectfully submitted in s... 阅读全帖 |
|
b****n
发帖数: 8 | 48 Address:
Email:
Cell phone:
Work phone:
Oct 28, 2013
Department of Homeland Security
USCIS Nebraska Service Center
P.O. Box 87103
Lincoln, NE 68501-7103
I-140 Immigration Visa Petition Under First Preference EB-1
Petitioner/Beneficiary: XX Z., Ph.D.
Category of Petition: Alien of Extraordinary Ability
Classification Sought: 203(b)(1)(A)
Nature of Submission: Original Submission
Dear Immigration Officer,
This letter is respectfully submitted in s... 阅读全帖 |
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m*******n
发帖数: 5103 | 49 此法案又称 GMO Labeling，于去年提出，在搜集足够签名后由州务卿咨请州议会表决
，但州议会并未进行讨论与表决。根据法律规定，未讨论的法案在第二年的普选时交由
选民公投，也就是今年的 11/5。如果通过，将在 7/1/2015 实施。
法案要求所有经过基因改造的食品，或者含有基因改造成分的食品，都必须明确标示。
正方认为这是民众知的权利，反方认为这会提高食品价格，尤其对本州的中小企业不利。
敬请有投票权的同学们关注此案，正反双方的意见请自行参考以下网站：
正方（主张必须标识）网站 http://yeson522.com/
反方（反对标识）网站 http://factsabout522.com/
阴谋论者可能会觉得反方一定是邪恶势力，但反方除了大公司以外，也包括本地
的中小企业。反对的原因除了提高成本，伤害本地经济以外，也有人说此法案本身不够
健全，如果实施，反而抓小放大，误导消费者。
重点是：天下事情没那么简单 --- 邪恶势力 vs. 屁民，正反双方的意见都有他们的道
理。建议同学们学习学习再决定。
此法案只是说基因改造的食品必须标识。至于基因改造的食品对人究竟有害无害，不是... 阅读全帖 |
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E********8
发帖数: 22 | 50 possible publication list for people graduated since 2010
118 伊成器
1. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-
associated FTO.
Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG
, He C.
Nat Chem Biol. 2011 Oct 16;7(12):885-7. doi: 10.1038/nchembio.687.
2.Targeting MgrA-mediated virulence regulation in Staphylococcus aureus.
Sun F, Zhou L, Zhao BC, Deng X, Cho H, Yi C, Jian X, Song CX, Luan CH, Bae T
, Li Z, He C.
Chem Biol. 2011 Aug 26;18(8)... 阅读全帖 |
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