g*********5
发帖数: 2533 | | H****s
发帖数: 301 | 2 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 problem. So be careful with the
trade offs. For example, if you use puromycin and your target gene does not
share promoter with puromycin resistance gene, there is good possibility
that some puromycin resistant clones will not express your protein of
interest. Even worse, if you use pooled clones, those non-expressing clones
will outgrow clones that express your protein of interest.
【在 g*********5 的大作中提到】
: thanks.
| q*****n
发帖数: 331 | 3 My personal experience:
neo and puro are good, quick selection.
Hygro is OK, but kill cells slowly.
Zeo is the worst, even zeo resistant cells have funny shapes. After a few
days of selection, you have to trypsinize cells off the plates and replated
cells without zeocin. Only zeo resistant cells will survive and grow. | g*********5
发帖数: 2533 | 4 非常感谢。我准备用两个lentivirus质粒转两个基因，so neo and puro. | z*******6
发帖数: 679 | 5 I don't think the selection marker matters a lot. I think it is the cell
line and the way of transfection that matters more. If you don't care the
expression level tooooo much, lentivirus works really good. But last time I
was doing A549 and I need the expression level to be really high to secrete
a lot of soluble proteins for ELISA detection, none of methods (
electroporation, nucleous transfection, lentivirus) or selection markers (I
tried multiple ones) worked. | g*********5
发帖数: 2533 | 6 how is the blast?
this marker work well? |
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