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研究者称成功重复韩氏 NgAgo 方法
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我虽然是外行看热闹，但字还是认识的。大家可以看看。该研究者称已经成功运用韩氏方法，目前正在进行基因测序确认。从讨论看，大家仍然在探索之中，而且并非完全重复韩论文，而是有所变化。
【We have used this system and it works. We have followed the same protocol as mentioned in the paper and were able to confirm knockout both on FACS as well as western. 】
该研究者 Dr.Debojyoti Chakraborty 履历好像比方舟子强一个量级或以上。参见《对韩春雨论文的质疑不应在暗处》。
Dr.Debojyoti Chakraborty
Post Doctoral Researcher [December 2013 – November 2015]
Max Planck Insitute of Molecular Cell Biology and Genetics & Technical University, Dresden,Germany
PhD in Molecular Biology, 2014 [Magna Cum Laude]
Thesis title: Systematic dissection of long noncoding RNAs involved in the regulation of embryonicstem cell pluripotency
German Stem Cell Network Travel Award, Frankfurt, Germany [2015]
German Stem Cell Network Conference Poster Prize, Heidelberg, Germany [2014]
Nucleic Acids Research Poster Prize, Heidelberg, Germany [2014
]Dresden International Graduate School Travel Award, Dresden, Germany [2012]
Future of Science Foundation Scholarship, Keystone Symposia, Utah, USA (2012)
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we have successfully used NgAgo in HeLa cells following a protocol where we used 700ng NgAgo plasmid and 500 ng gDNA (non purified, heat inactivated PNK). We confirmed knockout of GFP from a plasmid and could see the effect even after 96h. About 40% reduction in Western blot could be seen although there is scope for more optimization.
cheers
debo
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We have used this system and it works. We have followed the same protocol as mentioned in the paper and were able to confirm knockout both on FACS as well as western. We purified the oligos on gel after T4 PNK and saw the phenotype after 48 and 72 h.
cheers
debo
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We first phosphorylated the gDNA using the PNK kit from Ambion for 1 hr followed by heat inactivation at 95 degrees for 5 min. We then went on to transfect 100,000 cells in 12 well plates as follows:
Mix 1:
Lipo reagent- 3ul
Optimem- 43ul
EGFP N1- 1 ug
NgAgo- 900 ng
gDNA- 500 ng
Mix 2:
Lipo 3000- 2ul
Optimem- 48ul
Mix 3:
Media- 250ul
Optimem- 150ul
Mix1 and Mix2 was incubated for 10min each and then both were mixed together and incubated at RT for 30min
This was now added to cells. To each well, 50 ul of Mix 3 was added on top. No further media was added to the cells.
After 12h, the mix was removed and fresh media was added.
Just had a couple of tests with ngago from addgene and custom Oligos, so I did not try to reproduce the paper.
And in my hands I see an effect that is slightly less than using the same target sequence region using the px459, but it works.
I phosphorylated the oligos using T4PNK and transferred 300 ngago per 24 well to hek293t cells.
Cheers
Jan
https://blog.sciencenet.cn/blog-684007-988588.html
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