c********6
发帖数: 693 | 1 From Google 网上论坛
Michael Wilson
2016-07-15 16:54（1 分钟前）
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Hello,
We have twice attempted to test the NgAgO addgene plasmid in HEK 293 cells
using lipofectamine transfection and the EGXXFP split reporter assay. As
many are aware, this assay requires DNA cleavage in the EGXXFP plasmid to
restore GFP expression.
We ordered oligos with 5' phosphorylation and transfected directly or PNK-
treated an aliquot prior to transfection, just to ensure 5' phosphorylation;
neither of these methods restored GFP expression. In contrast, the positive
controls (Cas9 and gRNA expressed from pX330) gave robust GFP expression.
I will echo others here that there is a discrepancy between what the authors
have shown and what others have been able to repeat. I don't believe anyone
has reported indels yet, correct? | c********6
发帖数: 693 | 2 Genome-editing
下午4:50（6 分钟前）
其他收件人： h********[email protected], c******[email protected], o*****[email protected]
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hello guys,
we tried NgAgo plasmid cotransfection with 5'phospho-ssDNA guides and I must
say cell mortality rates are very high (toxic). We got 5'phospo-ssDNA
guides synthesised from IDT, desalt purification. Does anybody have tested
desalted verus PAGE purified ssOligos and looked for cell viability.
cheers! |
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