B******u
发帖数: 23763 | 1 找张纸，
俺们学校不在他们数据库里。
哈哈，这还是版丝建议的。
J Bone Joint Surg Am. 2009 Aug;91(8):1973-84.
Recombinant human platelet-derived growth factor-BB augmentation of new-bone
formation in a rat model of distraction osteogenesis.
Moore DC, Ehrlich MG, McAllister SC, Machan JT, Hart CE, Voigt C, Lesieur-
Brooks AM, Weber EW.
Orthopaedic Research Laboratories, Department of Orthopaedics, Warren Alpert
Medical School of Brown University/Rhode Island Hospital, CORO West Suite
404, 1 Hoppin Street, Providence, RI 02905, |
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z******g
发帖数: 1067 | 2 ☆─────────────────────────────────────☆
ngaido (ngaido) 于 (Sat Feb 28 10:48:34 2009) 提到:
中国古诗里我最喜欢的是唐诗，主要原因是觉得唐诗言之有物，里面有货，thought
provocative。杜甫很实在，李白最有想象力。比如月下独酌，从独酌，到和月与影成
三人，接着‘月既不解饮，影徒随我身’，。。。‘我歌月徘徊，我舞影零乱’，。。
。最后‘永结无情游。。。’，写尽孤独，不管是怅然还是潇洒，都很大气。再过几个
朝代中国的古体诗就大多是recycle和recombination.有自己original想法的很少。
至于什么是诗，poem，我是不知道明确定义的。也不知道在何处划界。这些都是专业人
士的研究争论节目。wikipedia，the encyclopedia for dummies,是这么说的。
Poetry (from the Greek "ποίησις", poiesis, a "making") is a
form of literary art in w |
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h**c
发帖数: 2376 | 3 几个月前呆大简简单单的一句“recycling and recombination”(R&R)，震动了我的心
。一直念叨到现在，真叫做“余音绕梁，三月未绝”啊！
呆大的确解决了让我疑惑了很长时间的问题。-- 有时候读近一点的古典诗词，明明挺
好的几句，放在一起不知为啥就显得是凑合，读多了味如嚼腊，没意思的很。-- 读清
朝的词的时候尤其明显。记得一个还是挺有名的词人，写的一首挺好的词。可我第一遍
读下来，脑袋里就一个反应 -- 这词我也能写！
现在看来，肯定是因为我在他的词中看到了太多前人的单词以及用法，觉得自己随便到
宋词里找找凑合下，也能胡诌出来一首的说。
那么如果喜爱旧体诗词，而不喜欢新体诗的话，怎样才能避免这种拾人牙慧的情况呢？
我觉得
一个选择是这个人是天才，就是recycling，也能比前人漂亮。比如曹雪芹说的王维的
“大漠孤烟直”是从陶渊明的“依依墟里烟”出来的，那前一句对我来说比后一句更吸
引人。
天才就是天才，能超越前人呀。
如果不是天才，又爱写点旧体诗，那该怎么办呢？我觉得还有一种方法，就是红楼梦（
以及
其他很多古典小说）里的那种，每一首诗都有它很详细的背景和含义 |
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y****e
发帖数: 71 | 4 If contamination goes 99.9% in all clone, that means you get a supercoiled
circular DNA as your contaminant. It transforms at a much higher efficiency.
First thing you might do is change plasmid.
I talked it to my boss, he had an interesting theory. If your insert confers
toxicity to E.coli, he said there might be some wierd recombination event that
cut the insert out of the plasmid, along with flanking reagions (which might
contain the MCS, that is why you cannot cut it again with your REs). |
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d****t
发帖数: 26 | 5 The following is from
http://www.nidcr.nih.gov/news/inside%5Fscoop%5Fcancer%5Frsch.asp
CANCER RESEARCH: KILLING TUMOR CELLS WITH ANTHRAX-BASED IMMUNOTOXIN
As the news broke last fall of anthrax-tainted letters, some reporters
eventually spun the story in a completely different direction. They
highlighted early research studies in which scientists use modified anthrax
proteins to fight human disease. Among the work mentioned prominently has been
studies at NIDCR to produce a recombinant immunotox |
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g****r
发帖数: 14 | 6 Kozak
Sequence for
Mammalian
Expression
If you will be recombining your entry clone with a
destination vector for mammalian expression, your insert
should contain a Kozak consensus sequence with an ATG
initiation codon for proper initiation of translation (Kozak,
1987; Kozak, 1991; Kozak, 1990). An example of a Kozak
consensus sequence is provided below. Other sequences are
possible, but the G or A at position -3 and the G at position
+4 are the most critical for function (shown in bold). The
AT |
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j********o
发帖数: 2 | 7 Yes. Not only BL21codon plus, but almost all BL21s except BL21(DE3)pLysS/E are
leaky. pLysS/E encode enzyme that degrade T7 polymerase to avoid leaky.
In your case with many rare Arg codons, you have to use codon plus. So, it is
importment to avoid leaky? otherwise, just express with leaky if it does no
harm. |
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s*******e
发帖数: 740 | 8 哦，忘了提醒你一下
关于第三点，一般人们并不用target序列做probe，而要把probe选在target区域外
这是因为你的序列也可以randomly整合到genomic DNA中去，而你只想要homologous
recombinate的产物，所以不要用target序列做probe，否则有很多假的positve
Good luck! |
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l*********i
发帖数: 332 | 9 1.DTA toxicity issues, i don't know how bad it will be, but may be better
than nothing. that is why NKCC1 provides another excellent target
2.the efficiency of nestin-cre-er to target neuro-progenitor in hippocampus
does matter.
3. hippocampus-loxP-GFP-loxP-DTA, based on transgenic, so need to select
single copy, in order to increase recombination efficiency.
and this is double transgenic strategy, oh, boy, i've got to say,
the less steps you are working on, the more success you may get about mo |
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x********u
发帖数: 430 | 10 We use T7 promoter for our vector system. We have been very successful to
produce the recombinant protein for bioconversion. Usually we induce the
cell with IPTG for 3-4 hours before bioconversion. But this time I induce
the cell for about 12 hours and I didn't get any products. The cell grows
well and seems normal. What's the consequence if you induce the cell too
long with IPTG? Does this matter with my expression results? Thank you! |
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s*r
发帖数: 2757 | 11 you are right. y-chr has no recombination, few mutation and low
heterozygosity |
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X***n
发帖数: 366 | 12 Anyone can help?
I need pKD46 to retrieve a genomic sequence from BAC.
Do you know which company sell pKD46? |
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X***n
发帖数: 366 | 13 Thank you very much!
I really appreciate your help! |
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h*********9
发帖数: 361 | 14 No, totally different. site/gene-specific insertion by homologous
recombination. |
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n********k
发帖数: 2818 | 15 it depends on what you are doing, and how big the risk u like to take?
Nobody really knows in practice(I
asssume u are doing mice knockout?......for easy targeting locus, it is
likely fine but if you are unlucky, u
might get nothing...if it is easy to correct, I would just remake it,
otherwise go ahead with your targeting and
see what happens...If it were me, I'll definitely remake the construct, it
takes nothing to remake one:))
compare to the targeting,. one step is about a week work, isn't it |
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n********k
发帖数: 2818 | 16 BTw, there are several experts on this board on the topic, maybe they will
have different opinion.... |
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j*****q
发帖数: 82 | 17 多谢ls的。
这100bp不到的序列是一定要加的，类似一个mutation。HR的目的是knock in。因为就
100bp不到的sequences，我就直接做在一个homo arm里了。不知道可不可以。
继续等大大 |
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n********k
发帖数: 2818 | 19 thanks a lot, don't have anything like that in planning , just something I
thought i shall ask your opinion about
it in case I need to do in the future...so far everything is on track,
finished one study last year and trying to
wrap another one this year and then hope to have more freedom to think about
moving on...
,
It' |
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j*****q
发帖数: 82 | 20 hi, henben,
yes, the less than 100bp contains a loxp site. acutally i am also aware of
two HR event in it. but i dnt have any marker to select the 100bp events.
but it is quite easy to distinguish the insertion.
does it mean the second HR to integrate the 100bp will be very low
efficiency?
does it mean the introduction of 100bp will be just by chance? |
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j*****q
发帖数: 82 | 21 and Happy new year all guys. |
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j*****q
发帖数: 82 | 23 3k+5k的位置。
does it matter？ |
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H****N
发帖数: 997 | 24 Sorry I did not see this earlier. You don't need a selection marker for the
insertion, but you need a genotyping strategy to distinguish the two HR
events. |
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s******y
发帖数: 28562 | 25 For yeast, it will be alright.
For mammalian system...I don't know |
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s********s
发帖数: 84 | 26 测基本上是，是把得到的virus稀释后 infect细胞 然后用抗生素blasticidin 选出来
有抗性的以后 染色 数有多少colony。
载体重组了。。如果是这个原因 有啥办法呢。。只能换载体么。。
to neverthink 更多的细节啊
是用PENTR3C （里面有表达目标蛋白的gene）
然后和 plenti6/V5-DEST 做LR recombination
得到colony做酶切 看看位点之类的对不对
最后得到plasmid （用来做transfection之类的能得到蛋白表达）
用这个plasmind然后和pLP1, pLP2, pLP/VSVG之类的一起transfection 293T 细胞
得到virus
然后infection 然后检测蛋白没有。。
曾经用同样的办法做了几批virus，曾经有一批是能检测出来的。
但是后来我再做就不行了= =
反反复复几次以后 老板就让我重做一遍vector 就是从PENTR3C和自己目标蛋白gene 酶
切，ligation那步开始重做。也不说为啥要这样。一摸一样的方法重做一遍会有啥改变
么。。
PENTR3C这个原始的载 |
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f********7
发帖数: 59 | 27 辩论如果把别的基因转到细菌里后，会不会产生超级bug.现在看来似乎不会。 |
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s******y
发帖数: 28562 | 29 你是想问Homologous recombination?
这个很简单啊，就是序列相似的DNA片断在重组酶的作用下发生互换。
其中涉及相似片断的结合，和交叉点(Holliday junction)的切断，
新片断的连接三个步骤。 |
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s******y
发帖数: 28562 | 30
Yes, but will happen in a lower probability. When there is DNA damage,
several signal pathway will activate the recombinase system and increase the
recombination frequency.
What do you mean ? I cannot understand your question.
If you are wondering if we would put a homologous region in the designed
fragment in order to target it to a specific region, then the answer is
"YES, we do".
If you want to know the theory and practice of doing knockout, the easier
way to to look at wikipedia |
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g***y
发帖数: 201 | 31 多年没做这个了，知识比较老化。
1.比较两个arm的sequence homology between 129 and B6.
一般的担忧是因为两个strain的序列有差别，可能影响同源重组的效率。但是如果幸运
的话也许你感兴趣的区域两者高度同源。
否则，几个OPTIONs
A。选用B6 derived ES CELL,有些service提供的。
B.让你的arm尽量长. 但是还是不能保证一定会有positive clone,要看运气，并且问题
是plasmid大到一定程度就不稳定了。比较safe的办法是用BAC recombineering做
BAC based construct.
C。 use long-range high fidelity DNA polymerase to PCR 129 genomic sequence.
这个对cloning要求有点高，并且要做sequencing确定没PCR error |
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r**h
发帖数: 6 | 32 谢谢指教。
找不到129的BAC，也许意味着129的这个位点没有测序，所以看来比较难比较两个
strain的序列。我自己也许可以PCR出来，自己测，不过感觉事倍功半。我想当然的比
较多，还请指正。
比较遗憾我们使用的service质量不算上乘，做B6 derived，有点战战兢兢。LOL...
谢谢指出这一点。我准备用recombineering，所以希望有BAC。但是如果找不到，就只
能PCR 129 arms，然后一步步酶切连接了。
sequence.
非常感谢你的答复。 |
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a******u
发帖数: 211 | 33 J Immunother. 1998 May;21(3):159-69.
The use of a cationic liposome formulation (DOTAP) mixed with a recombinant
tumor-associated antigen to induce immune responses and protective immunity
in mice.
Bei R, Guptill V, Masuelli L, Kashmiri SV, Muraro R, Frati L, Schlom J,
Kantor J.
Please send the paper to e*********[email protected]
Thanks so much!! |
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I*****y
发帖数: 6402 | 34 要买recombinant CDK kinase enzyme, 最好是Xenopus or human的。请大家推荐一个，
做kinase assay |
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w******e
发帖数: 1187 | 35 有没有价格便宜量又足的地方？要用很大量的protein，查了下millipore，每
25ug 300刀，太贵了。。。 |
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e***l
发帖数: 33 | 41 发个小广告，投条给我
Positions: research associates 1 and 2 (RA1, RA2), production associates 1
and 2 (PA1, PA2).
Location: Frederick MD. (no relocation package)
Company: top tier in biotech
Degree: BS/MS + 0-4 years experiences
Area: immunology assay development, (or) mammlian cell culture, (or) recombinant
protein/antibody purification
Has huge career development potentials.
Has very specific requirement. So don't feel sad if I tell you it doesn't
fit you. |
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X***n
发帖数: 366 | 42 I cloned 400-500 bp miniarms cloned into ptdTomato-C1, and then linearized
it with EcoRI, so that the backbone was flanked by miniarms on both ends. I
electroporated the linearized plasmid into the induced Red recombinases in
SW102 carrying my BAC. All the clones turns to be empty. I succeed in the
other two using pKD46 system but failed in this one. And now I also fail
using SW102 system. What's the most possible reason? Thanks in advance. |
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p**i
发帖数: 3525 | 43 每次提纯抗体 (全部) 需要多少可溶解的recombinant protein和beads?
us
. |
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L**********y
发帖数: 499 | 44 Do recoembination. It is very simple to recombinate your UAS and tub-Gal4
into the same chromosome. |
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x**w
发帖数: 112 | 45 homologue recombination |
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o**i
发帖数: 1165 | 46 你要想不让cre 去binding 还是不去recombine
你不让cre去work 你干嘛放loxP上去
mutation/
both |
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h***e
发帖数: 146 | 47 我也不是啥牛，我个人观点，现在比较fashion的是 1,in vitro reconstruction &
chemoenzymatic biosynthesis. 2 是 组合生物合成
至于技术： 基本上是建库，基因克隆和修饰的那些技术（貌似 Red/ET
recombineering用的挺多），还有蛋白纯化，结晶等等。
我认为火可能是因为与药物联系紧密，还能产生一些化学合成难以达到的衍生物。另外
研究生物合成机理本身也是很奇妙的，一些酶催化的反应也很interesting。
恩， 我发现我也是外行 |
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J*****u
发帖数: 15 | 48 Methods Mol Biol. 2009;521:345-59.
Isolation of recombinant DNA elongation proteins.
Email:j******[email protected]
Thanks |
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p*******r
发帖数: 4048 | 49 Have you tried recombination incompetent bacteria? |
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