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F******p
发帖数: 2099 | 2 first do it on plasmid, then recombinant it into genome. |
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h**********r
发帖数: 671 | 3 Title: Recombinant Expression in Moderate Halophiles.
curr pharm biotechnol 11(3):259-66 (2010)
Author: Masao Tokunaga, Tsutomu Arakawa and Hiroko Tokunaga
E-mail: x*****[email protected]
Thank you very much! |
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j*****a
发帖数: 92 | 4 拿有我insert DNA的recombinant plasmid(pRSet A)去测序，１００ ％ 符合。（我的
insert有1.０ kb），transform into BL21(DE3)pLysS Competent Cells 用His柱子纯
化蛋白. SDS-PAGE显示分子量20 kDa, 应该是35 kDa. 谢谢高手们回答。 |
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j*****a
发帖数: 92 | 5 谢谢大家的分析。我的insert DNA是N-His tag。 如果是翻译提前终止或降解，有什么
方法补救吗？
背景：拿有我insert DNA的recombinant plasmid(pRSet A)去测序，１００ ％ 符合。
（我的insert有1.０ kb），transform into BL21(DE3)pLysS Competent Cells 用His
柱子纯化蛋白. SDS-PAGE显示分子量20 kDa, 应该是35 kDa. |
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X***n
发帖数: 366 | 6 有BAC clone 吗？有的话基于BAC clone recombineering delete掉不需要的部分就行
了。
PCR clone是够呛的，基因组不大的话自己做BAC未必不是个选择。 |
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h***e
发帖数: 146 | 7 我估计用recombineering不好，repetitive segement太多 |
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P****d
发帖数: 564 | 8 据说recombinant DNA是Stanford一个graduate student写qualify proposal时编的一
个fake project，结果几个committee members赶回去做实验 ...
又据说，Maxam-Gilbert测序法是Gilbert另一个学生提出来的想法，但是觉得没意思，
没做。我倒是认识这老兄，不过从来没敢问过。 |
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a***e
发帖数: 1010 | 9 我的前老板说 recombinant DNA tech 是她在 stanford 做的。 |
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h***e
发帖数: 146 | 11 Red/ET recombination really helpful....... |
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i*********0
发帖数: 915 | 12 用Recombineering，或者直接按照传统的做法，pcr，长臂，短臂。 |
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a****k
发帖数: 1130 | 13 如果是recombinant protein的话，最好加
如果是cell extract的话一般不用加，protein concentration高的时候不太会出问题 |
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a***e
发帖数: 1010 | 15 (1) for any model organism, you can try homologous recombination to mutate
anything you want.
(2) for model organism like yeast, worm, fly, the best part of them is
genetics. You can do "unbiased" genetic screen to identity anything change
a defined phenotype. |
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O******e
发帖数: 4845 | 16 1 ug of recombinant IFNg has about 104 units. So 15U/mL is about 0.144 ug/mL. |
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c****l
发帖数: 1086 | 17 即使在同一条染色体上还可以发生homologous recombination而组合在一起啊。
只是不能太近，这个几率可以用cM来测量。如果是在同一染色体的两头，重组的几率还
是不低的。
The centimorgan is equal to a 1% chance that a marker at one genetic locus
on a chromosome will be separated from a marker at a second locus due to
crossing over in a single generation
One cM is about 1 Mb apart. |
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l********s
发帖数: 5 | 18 Recombinant Factor C competes against LBP to bind lipopolysaccharide and
neutralizes the endotoxicity
J Endotoxin Res. 2007;13(3):150-7.
l************[email protected]
谢谢! |
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f******s
发帖数: 288 | 19 恩，前一阵还有一个影响也很大，pfizer/Wyeth 的 Mylotarg ，这是fda 批准的第一
个immunoconjugate， 好像也是迄今唯一一个，而且是第一个通过accelerated-
approval 批准的药 ，2个月前宣布不能prolong survival ，只好withdraw。现在很多
药厂／生物公司都有一批在pipeline上的immunoconjugates，所以还是让不少人讨论了
一下的。
不过说其中药，最大的问题是无法控制活性成分，还有产地条件，原料都是有很多不可
控的变化的。所以，我总觉得，在某种程度上，中药根生物制品还是很像的。。。不可
控因素很多，没有足够的检测方法，很多时候无法设定specification是什么。
其实最初的疫苗，血液制品都曾因为质量／来源 控制的问题带来了很多灾难，直到
recombinant protein 出现了，质量控制才前进了一些，但是仍然问题很多。。。
恩，还有最近好像确实有个中药在美国做的phase ii，据说要开始phase iii 了。 |
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s******y
发帖数: 28562 | 21 Yesh, for example you can use IP on endogenouse proteins
For the protein domain things, you can use the recombinant domain of A to
pulldown the endogenous proteins B |
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b***4
发帖数: 12 | 22 凯因科技 (www.kawin.com.cn)
Biotech company
- Recombinant protein drugs/vaccines
- Not founded by returnees, but has a very capable and open-minded CEO
- >100MM RMB revenue
- Plans to go public in three years
Looking for a R&D head to lead
- due diligence for in-license products
- new candidate development
Criteria
- Ph.D. in cell biology, molecular biology, pharmacology or related field
- Good understanding of mechanisms of biotherapeutics and drug
development
- Industrial |
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H****N
发帖数: 997 | 23 How was the KO made, by homologous recombination or gene trapping? Why is
there cre in it? If the mouse is made by gene trapping, it is possible the
gene is not knocked out completely, depending on where the gene trapping
cassette is located. Another remote possiblity is that there is another
related or duplicated gene in the genome, and thus your results. |
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h*****y
发帖数: 10 | 24 It's a homologous recombination deleting one exon. And this gene is a member
of protein family. |
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a*****g
发帖数: 543 | 25 what kind of Cre did you use?
If you happened to have SOX2-Cre, you will be able to get good KO( of target
ed site).
I'm assuming you are doing appropriate control of the genotyping, with both
WT primers and recombinant primers...
How big was the targeted exon? where is your qRT-PCR targeting? Having one p
rimer locating in the exon may help to clarify whether deletion of the exon
occurred... |
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b***4
发帖数: 12 | 26 炒旧帖，诚聘研发总监
凯因科技 (www.kawin.com.cn)
Biotech company
- Recombinant protein drugs/vaccines
- Not founded by returnees, but has a very capable and open-minded CEO
- >100 million RMB revenue
- Plans to go public in three years
Looking for a R&D head to lead
- due diligence for in-license products
- new candidate development
Criteria
- Ph.D. in cell biology, molecular biology, pharmacology or related field
- Good understanding of mechanisms of biotherapeutics and drug
developmen |
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o********r
发帖数: 775 | 27 homologous recombination + positive/negative selection?
不过俺N年没做过这个了。。。 |
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v*******8
发帖数: 12 | 28 我需要这段基因由细胞内某endogenous promoter来调控，所以应该要插入在特定位置
不是简单的recombination |
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a*****y
发帖数: 277 | 29 You are right. When GFP is in the periplasm it is not folded properly.
However some outer membrane, when expressed recombinantly, can be in both
inner membrane and outer membrane, in which case you'll see GFP Fluorescence
.
There are certainly other fluorescent proteins - I can not remember the name
now. it is called something like ilov or ilove or ilike...? |
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b****n
发帖数: 311 | 30 from wiki
During the late 1970s, Axel, along with microbiologist Saul J. Silverstein,
and geneticist Michael H. Wigler, discovered a technique of cotransformation
, a process which allows foreign DNA to be inserted into a host cell to
produce certain proteins [2]. Patents, now colloquially referred to as the "
Axel patents", covering this technique were filed for February 1980 and were
issued in August 1983[3]. As a fundamental process in recombinant DNA
research as performed at pharmaceutical a... 阅读全帖 |
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c***b
发帖数: 12 | 31 Science. 1994 Sep 2;265(5177):1442-5.
Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J
recombination.
Taccioli GE, Gottlieb TM, Blunt T, Priestley A, Demengeot J, Mizuta R,
Lehmann AR, Alt FW, Jackson SP, Jeggo
PA.
Howard Hughes Medical Institute, Children's Hospital, Boston, MA.
Link: http://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=8073286
please email to h*******[email protected]
thank you very much |
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T**********t
发帖数: 1604 | 32 我没做过chromatin，所以说得不对的话请大家批评。
我主要是做recombinant protein expression比较多，在我用的protocol里，lysate
sonicate之后，高速离心之前的一个步骤就是加4%的PEI (polyethyleneimine)到终浓
度为0.2%左右。这一步就是为了去除DNA的，我做过gel shift，PEI处理过的蛋白跑的
胶确实没有DNA，而不经PEI处理的蛋白就会有DNA contamination，区别还是很明显的
。我的lysis buffer里没有加DNase，貌似PEI的效果就很好了。
但是不知道PEI对你的样品和后续处理有没有影响，所以只是作为参考。 |
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A***a
发帖数: 144 | 33 Invitrogen 怎么会没有卖？
Insulin, Human Recombinant, Zinc Solution 5 ml
Cat. No. 12585-014 |
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h***e
发帖数: 146 | 34 建库 然后用red/ET recombineering 来stitch ？ |
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e***o
发帖数: 344 | 35 Pred/ET recombineering ,
easy ! |
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t********7
发帖数: 22 | 36 谢谢！ 我想克隆的60kb gene islands 在某一bacteria的genome里，请问能推荐什么好的PCR方法可以一次把它整片段都PCR出来，
是不是只有这样有了linear insert之后，才能用pRed/ET recombineering把60kb插入到BAC plasmid里, 最后transform到另一个strain里？
不好意思，问题可能比较弱....不过真的不知道有什么好办法 |
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h***e
发帖数: 146 | 37 60KB作为insert 在 Red/ET recombineering 也实在太大啦啊
PCR还要测序 不保险 还是建库吧 然后用Red/ET 来 缝合 |
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t********7
发帖数: 22 | 38 不知道各位能不能建议一些建库和Red/ET相关的文章，这样我可以知道具体怎么操作和
可行性？
另外gateway需要需要有特定的att次序对来recombination，不知道Red/ET的也是一样
？还是可以任意的次序，只要是相同就可以？
非常谢谢！ |
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h***e
发帖数: 146 | 39 Volume 12, Issue 3, March 2005, Pages 349-356 Chemistry&Biology
Heterologous Expression of a Myxobacterial Natural Products Assembly Line in
Pseudomonads via Red/ET Recombineering
Silke C. Wenzel1, 2, Frank Gross1, 2, Youming Zhang3, Jun Fu3, A. Francis
Stewart4 and Rolf Müller1, 2,
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VRP-4FSV963-G&_user=1592387&_coverDate=03%2F31%2F2005&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1525352222&_reru... 阅读全帖 |
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p*****m
发帖数: 7030 | 40 我猜她说的是northern，这样才谈得上看什么3'转录的问题 对于KO来说看看northern
，或者对于转基因来说看看southern，而不光是PCR 主要还是考虑到KO可能会在recombination的时候发生了怪异的重组 甚至把切出来的片段插到别处去了 这一步因为有个open end在那里飘着 可能会发生没法预测的事情。transgene也一样，transgene还存在个copy number问题
southern |
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m******5
发帖数: 1383 | 41 这篇文章从头到尾都是在消化道中检测啊，而且不光是transgenicDNA，还有植物内源
DNA
文章主旨就是说外源DNA会survive到消化道尾端，并且被肠上皮吸收和消化
而这种消化方式 ‘was with no evidence to suggest that recombinant DNA would
be processed in the gut in any manner different from endogenous feed-
ingested genetic material’
民科们能不能花钱请人把文章读懂了再出来吠啊 |
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r***e
发帖数: 2539 | 42 Mol Imaging. 2003 Oct;2(4):297-302.
Mouse reporter strain for noninvasive bioluminescent imaging of cells that h
ave undergone Cre-mediated recombination.
发到
reuseisgood在yahoo.com
附上Mitbbs ID，方便我发包子。
谢谢！ |
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n****r
发帖数: 37 | 43 急用
多谢拉
Simultaneous expression of glutaryl-7-aminocephalosporanic acid acylase gene
and lysis genes of phage λ in a recombinant E. coli
麻烦发到m*****[email protected] |
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m*****n
发帖数: 5245 | 44 我的邮箱： m*****[email protected]
Kim HL, Seligson D, Liu X et al (2005) Using tumor markers to predict the
survival of patients with metastatic renal cell carcinoma. J Urol 173:1496–
1501
Mancuso A, Sternberg CN (2005) New treatments for metastatic kidney cancer.
Can J Urol 12(Suppl 1):66–70
Motzer RJ, Mazumdar M, Bacik J et al (1999) Survival and prognostic
stratification of 670 patients with advanced renal cell carcinoma. J Clin
Oncol 17:2530–2540
Fyfe G, Fisher RI, Rosenberg SA et al (1995) Results of... 阅读全帖 |
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s********n
发帖数: 2939 | 45 一些补充：
native protein or recombinant protein? 功能当然是首选；但如果有tag（如His
tag）就用其抗体检测，没有就当我没说。
首先还是功能检测，在不同温度下incubate不同时间然后测功能；DSC是测Tm的gold
standard
也可以用gel staining |
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s***e
发帖数: 911 | 46 1. 这个重组过程, 一定是在B-form dsDNA和RecA-ssDNA之间完成的吗?
2. RecA也可以直接和dsDNA作用, 形成dsDNA-RecA triplex. 这个结构在重组过程中有
任何作用吗? 比如, 重组可以不可以在dsDNA-RecA 和一个空的ssDNA之间发生?
3. 重组可不可以在dsDNA-RecA和ssDNA-RecA之间发生?
谢谢. |
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l*****u
发帖数: 11 | 47 好像公司卖的thrombin都是从plasma 提纯的
不知道有没有用recombinant 的方法自己制的
如果有的话， 你的plasmid 从哪儿来?
能否给个出处文献
多谢 |
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c**a
发帖数: 94 | 48 就是Histone acetylation/deacetylation,蛋白放一起做in vitro assay. 有没有什么
trick啊？买来的recombinant protein都不管用，positive control都做不出来。不知
道到底
哪里出了问题。 |
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f*********t
发帖数: 143 | 49 Thanks a lot in advance. Please send the paper to z*****[email protected]
Zebrafish. 2010 Jun;7(2):199-204.
Photoactivation of the CreER T2 recombinase for conditional site-specific
recombination with high spatiotemporal resolution.
Sinha DK, Neveu P, Gagey N, Aujard I, Le Saux T, Rampon C, Gauron C,
Kawakami K, Leucht C, Bally-Cuif L, Volovitch M, Bensimon D, Jullien L, Vriz
S.
Ecole Normale Supérieure, Laboratoire de Physique Statistique, UMR 8550
CNRS, Paris Cedex, France. |
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r**o
发帖数: 212 | 50 细胞培养用的是Sigma的牛胰岛素（insulin solution from bovine pancreas）,但是
现在Sigma缺货，backoeder不知道什么时候才能到货。转用Sigma的合成胰岛素（
recombinant from Saccharomyces cerevisiae）,细胞生长状态变差，出现凋亡状态。
多次从冷冻管活化，生长不到一周又会变差。
求教：不同来源的胰岛素对细胞生长影响那么大么？
有无其他公司售卖的牛胰岛素？
或者用Sigma的牛胰岛素powder替代，唯一的考虑是powder必须用酸性溶液溶解（
dilute acetic or hydrochloric acid, pH 2-3），是否影响细胞生长？原来的牛胰岛
素溶液是现成的溶解在25mM HEPES,pH 8.2里面。
困扰两个月了，求教各位达人了。多谢多谢！ |
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