n*****l
发帖数: 38 | 1 本人使用过多个基因的Morpholino进行knockdown处理,最近发现其中一个基因,我先配
制2mM的浓储,再稀释成1mM后进行测量(用nanodrop)发现2mM的才有1.15mM,1mM的才有0.
58mM.发邮件问客服,客服说我在配制2mM浓储的时候还有残留,但是我算了一下,如果有
残留,那就得有近一半的残留.可能吗?Morpholino极易溶解,我很确认分装的时候把液体
全部吸干净了.不知道哪位同仁遇到相同情况,进来讨论一下.谢谢! |
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s*******t
发帖数: 7746 | 2 SiRNA is NOT specific.
use Morpholinos oligos (made by Gene Tools,LLC, http://www.gene-tools.com/). It is antisense oligo with high specificity, stable in the cells (can not degraded by RNAase or DNAase or Proteinase etc). I have used Morpholinos in cell culture and mouse. Work really well. |
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W*********n
发帖数: 775 | 3 Thanks for the useful information.
For I want to use morpholino to block the traslation of the gene, not for
the splicing block, I think I know the 5'UTR and the starting 25bp coding
sequence--it is also required by the Morpholino company.
Thanks
a
for |
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W*********n
发帖数: 775 | 4 sorry for my misunderstanding.
Yes, you are right, I need to find the Translation Start Site or region
around CDS starting point.
Now, I have found the Tranlation Start Site, and also I can make sure the
downstream coding sequences after the TSS. So now I need a comfirmed 5'UTR
sequence, at least some of the sequence near the TSS. Do you think the
unstream of the TSS in the gemome is 5'UTR ? or at least we can make sure of
a samll seuquence before the TSS?
I seed the Coding sequence and 300bp up... 阅读全帖 |
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s*******t
发帖数: 7746 | 5 试试antisense morpholino oligo ,发育学斑马鱼等都用morpholino同时knock down几
个基因 |
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x**w
发帖数: 112 | 6 Eric Davidson
reductionism 的死忠拥趸
Gene tools的最有价值客户 (他实验室至少订过几千条 Morpholino)
不知道什么时候他画的那些“电路图”能在数学上被解释。。。
发信人: ppri (ppri), 信区: Biology
标 题: Re: wnt 通路还有得做没？
发信站: BBS 未名空间站 (Sat May 1 13:45:58 2010, 美东)
他到底有多牛? |
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s******s
发帖数: 13035 | 7 Wages JM Jr; Wages GM; Matthews P; Weller D; Summerton J. Affinity purificat
ion of RNA: sequence-specific capture by nonionic morpholino probes. Biotech
niques. 1997 Dec;23(6):1116-21.
或者有其他的用RNA pull down RNA的protocol,
请email z****[email protected]
多谢 |
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x***m
发帖数: 184 | 8 为什么在frog和chicken里面做genetic screen但在zebrafish里可以呢? 记得很多年前
就读到过用EMS突变老鼠进行genetics screen啊.
记得zebrafish也有genome duplication, 所以在downregulate gene expression上很
依赖morpholino. |
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p*****m
发帖数: 7030 | 9 basically the only thing you can do if morpholino. No RNAi, No good knock
out (the ZFN does not work for every gene); no good conditional KO, no KI... |
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c*********r
发帖数: 1312 | 10 Morpholino算不算一种啊？
我觉得LNA似乎也可以，和mRNA结合后很稳定，但是没见到过报道，它家网站上也没有
此应用。另外两种不了解。。。
如果试了管用告诉我们一声啊。^_^ |
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c*********r
发帖数: 1312 | 11 深刻！
这么说的话，specific translational blocking Morpholino如果能避免和microRNA重
叠的话是比较理想的knockdown的手段了。。。 |
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W*********n
发帖数: 775 | 12 想获取一个一个物种的某基因A的序列，从其5'UTR及开始的25个编码区碱基设计
Morpholino序列用于基因阻断。 从朋友那边（正在做此物种测序）获取了一包含这个
基因的测序序列：包含这个基因的所有内含子，外显子以及启动子区域 （只是中间也
有几个大片的NNNNN区）。我通过将其和另外一个亲缘最近的同属的其他物种（已经测
序）进行比较，找出了编码区的内含子和外显子（因为蛋白序列高度相似）。
现在的问题是：怎样从原始序列中找出5'UTR的确切序列？ 这个5'UTR是否一定在第一
个编码区的外显子上，还是可能在这个外显子的上游还可能有一个外显子，用来剪贴出
全部或者部分5'UTR？ 怎样判断5'UTR的起始位点？
请问用什么软件可以分析？谢谢！ |
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K**4
发帖数: 1015 | 13 I remember that some Japan groups have sequenced all UTRs of the genes and
transcript isoforms that generated by alternative splicing. They published a
lot of Nature and NG papers from this.
Dont know if they have made it public.
Go back to your purpose, I think you need to find TSS or splicing sites for
morpholino, not 5UTR, right? |
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s*******t
发帖数: 7746 | 14 不行，因为morpholino是作用在RNA上 |
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A******y
发帖数: 2041 | 16 Unless they did KI/KO using the new nuclease technology. Morpholino
injection is kind of old. |
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x**2
发帖数: 255 | 17 I think TALEN was used more than morpholino in that lab. |
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