h******y
发帖数: 351 | 1 http://genome.ucsc.edu/cgi-bin/hgBlat?command=start
Put your sequence in FASTA format, for example
>primer1
AGCTACAGCTACTAGCATCGACTGCGATG
>Primer2
ATGACTAGCTGGATAGCTAGCTACGATCA
>primer3
...
Make sure the primer sequence is longer than 20nt. So far this is the faste
st and most efficienct way I know. You can easily find out where the primer
s locate and whether there is any non-specific binding sites.
For pimer sequence shorter than 20nt, create four sequences by adding [AGCT]
to make it to 20n... 阅读全帖 |
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发帖数: 1 | 2 Email on ngAgo sent to the ISTT mailing list：
Dear colleagues,
the publication by Gao et al in May in Nature Biotechnology http://www.ncbi.nlm.nih.gov/pubmed/27136078 triggered an enormous expectation. This Chinese team led by Chunyu Huan reported that the Argonaute (Ago) protein from a rare haloarchaea, Natronobacterium gregoryi, (NgAgo) would efficiently work for gene editing purposes in human cells. Ago had been described as an DNA-guided endonucleases two years before, through a Dutch-Span... 阅读全帖 |
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z*t
发帖数: 863 | 5 他们有没有做miRNA直接的喂养实验？20nt的RNA synthesis起来应该不难 |
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m*********D
发帖数: 1727 | 6 看了IDT的website,他们的G-block合成，两个arm之间之容许20nt的随机sequence。而
我的有60-70nt。只自己克隆了。：（。
谢谢！ |
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m*********D
发帖数: 1727 | 7 简单地说，就是ZFN和 TALEN利用蛋白质上AA和DNA的Nt的相互作用来识别DNA的序列,一
般识别的长度在9-18bp。它们往往是两个domain--一个用于DNA序列的识别，一个是切
割双链DNA。
CRISPR/Cas是一个蛋白-RNA complex.RNA用配对来识别特定的DNA序列，蛋白用来切割
双链DNA。识别的长度在20Nt。
切割完后，产生double strand break(DBS)。修复都靠细胞里面自己的系统。在有修复
模板（可以人工提供）的情况下，找模板来，你要提供就可以按你的设计引入mutation
等等；没有模板，细胞就自己连接两头，顺便会插入/删掉几个bp（indel)，这就导致
reading-fram-shift,基因失活。
CRISPR就目前来看，操作容易，识别的特异性高（长度大）。估计会取代前两个。
你可以详细读这篇：ZFN, TALEN, and CRISPR/Cas-based methods for genome
engineering
http://www.sciencedirect.com/science/article/pii/S... 阅读全帖 |
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H****N
发帖数: 997 | 8 Dear colleagues,
the publication by Gao et al in May in Nature Biotechnology http://www.ncbi.nlm.nih.gov/pubmed/27136078 triggered an enormous expectation. This Chinese team led by Chunyu Huan reported that the Argonaute (Ago) protein from a rare haloarchaea, Natronobacterium gregoryi, (NgAgo) would efficiently work for gene editing purposes in human cells. Ago had been described as an DNA-guided endonucleases two years before, through a Dutch-Spanish microbiologist collaborating team (Swarts ... 阅读全帖 |
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发帖数: 1 | 9 【 以下文字转载自 Military 讨论区 】
发信人: hanchunyu (), 信区: Military
标 题: 韩春雨是第二个小保方－业界专家的信
发信站: BBS 未名空间站 (Fri Jul 29 11:59:57 2016, 美东)
Email on ngAgo sent to the ISTT mailing list：
Dear colleagues,
the publication by Gao et al in May in Nature Biotechnology http://www.ncbi.nlm.nih.gov/pubmed/27136078 triggered an enormous expectation. This Chinese team led by Chunyu Huan reported that the Argonaute (Ago) protein from a rare haloarchaea, Natronobacterium gregoryi, (NgAgo) would efficiently work for gen... 阅读全帖 |
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发帖数: 1 | 10 国际转基因技术协会原主席Montoliu博士建议不要再试图重复韩春雨实验结果
澳大利亚国立大学Gaetan Burgion今天撰写长文介绍他试图重复河北科大韩
春雨创立的NgAgo基因编辑技术的经过，得出结论：韩春雨的实验无法重复，而
且与韩在论文所说矛盾；《自然·生物技术》应要求韩公布原始数据；该技术显
然没有前途。
国际转基因技术协会创建者和原主席Lluis Montoliu博士根据这一结果以
及其本人实验室的实验结果，向国际转基因技术协会会员发出一封公开信，建议
停止继续试图验证韩春雨的实验，不要再浪费时间、金钱、人员和项目。
CRISPR谷歌群针对这项技术和韩春雨的文章的调查表明，140个调查回复中，
只有一个（中国神经所仇子龙？）回答该技术起作用，73个回答无效，63个回答
在验证。
From: [email protected]/* */ on behalf of Lluis
Montoliu To: ISTT List
Subject: [ISTT_list] Great disappointment with Ago: Long life to
CRISPR... 阅读全帖 |
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发帖数: 1 | 11 It's an interesting question. To the best of my knowledge, no RNA-seq
alignment tool was designed to tolerate CRISPR-mediated indels to date. I'm
not an RNA-seq expert, so it's possible that there are some on the way. You
can also email Luca Pinello, the author of CRISPResso. He might know more,
and he's a really nice guy.
Here I'm trying to think about a solution. There are two situations - I don'
t know which one is your case.
a) The sample for RNA-seq was derived from a single mutant clone. I... 阅读全帖 |
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