b******9
发帖数: 425 | 1 做了个construct，转primary newborn astrocytes，用hygromycin来筛选stable
transfection，可是一直都没有筛出来，细胞全都死光光了。转染效率还算可以，是电
转，有30%左右。hygromycin浓度也不算高，50ug/ml。
hygromycin是用TK promoter表达的，不知道是不是因为promoter不行。试了别人的一
个puromycin construct，是pGK promoter，也基本上死光了。想问一问版上牛人有没
有经验可以传授。多谢多谢！
另外我是用CMV promoter来表达我要的基因，先转细胞，然后筛选stable line打到老
鼠上。听说CMV promoter在体内可能会被shut down，是不是也应该换掉呢？谢谢！ |
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c****y
发帖数: 14 | 2 Hi, zgrzfg (wushi),
You think that
1) high expression of your gene is toxic to cells and in long-tern culture
these cells gain growth disadvantage against those with lower expression,
and eventually will lost expression of transgene, even under selection
pressure;
Actually I cotransfect the plasmid and Hygromycin marker plasmid into
tetracyclin-inducible HeLa cell line and got stable cell line by selection
with hygromycin. I still didn't detect protein expressed by western blot in
such stable c |
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h******y
发帖数: 351 | 3 Hygromycin浓度偏高了。你有没有先做一下kill curve找出合适的antibiotic浓度？
合适的hygromycin通常在10-30 ug/mL
合适的puromycin通常在0.3-1 ug/mL |
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s*****g
发帖数: 7857 | 4 test transfection efficiency没有做.因为他们网上有我细胞系的program参数。大约
在60%
用的什么antibiotics做的selection -Hygromycin （通常你们的细胞系都要多少浓度
？）我是用MTT来测titration的。
是否是由于四细胞和活细胞在一起，死细胞对活细胞有影响，而造成的呢？ |
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C******r
发帖数: 790 | 5 我拿到的BAC，没转那个带有recombinase的低copy质粒的时候，就能在hygromycin里长
了。。。
现在这个课题已经由一个学生接过去了，伊也很郁闷的在整，不知道伊新搞来的BAC好
用不好用。
tag |
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H****s
发帖数: 301 | 6 It depends on your purpose and the cell line to be transfected. Generally
speaking, if you cell line is tough enough, puromycin is much easier to work
with. You just need to add a relevant amount of puromycin to the media and
it starts killing.
For neomycin and hygromycin, you have to determine optimal killing
concentration specific to the cell line you will use. To initiate killing,
you have to split cells and supplement with neo/hygro containing media.
Every selective marker has its specific p... 阅读全帖 |
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g*********5
发帖数: 2533 | 7 求lentivirus质粒pLEX MCS
hygromycin resistant?
公司discontinue了，不知道能不能求到。我有些质粒可以交换。 |
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b**********8
发帖数: 349 | 8 最近要做一个inducible shRNA 的stable transfection，采用的细胞株之前已经做过
一遍稳转，G418 resistance。现在改用Hygromycin B筛选新的稳转株，已经挑了将近
100个克隆了，一个阳性的都没有挑到，很郁闷。想问下有经验的战友们，已经做过一
轮稳转的细胞株，再做新的稳转，是不是难度特别大？什么原因呢？难道是我人品太差
？ |
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e**r
发帖数: 1144 | 10 包retrovirus用的Phoenix细胞系
作者建议定期用DT+Hygromycin筛选
有什么筛选标记基因可以抗DT吗？ |
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发帖数: 1 | 11 For those who can not access the link
I copied the content here:
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
NgAgo
15 posts by 9 authors
Davide Seruggia
Jun 23
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
k*****[email protected]
Jun 24
Hi, Davide,
I tried th... 阅读全帖 |
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