Dilution cloning of HEK293
hubahopp at gmx.de
Tue Apr 27 17:27:11 EST 2004
of course, they don't like it. They feel as lonely as humans you put into
96 well plates. But normally, it works and by limited dilution, you have
much less hassle than with cloning cylinders.
You did not tell us how long you wait for your cells to grow, but if you
expect one cell to divide once or twice a day, it still lasts quite long
until you really can *see* a clone. In my experience with balb 3T3MF, it
may last more than one week or even more, especially if you select stable
transfectants with antibiotics. And a small cell lump of 100 cells is still
difficult to find with a microscope. If you can see a change in the media
redox indicator color, it's time to have a closer look.
You might use conditioned medium (eg mix supernatant of confluent cells
previously sterile filtered with fresh medium 1:1) to make them grow a
little faster and feel happier.
Before you seed the cells into microwells, you could perform the antibiotic
selection in an ordinary dish to find out if you get stable transfectants
at all (and to get rid of not transfected cells). Then you have only cells
expressing the marker you can use for clonal selection. And you don't need
to screen so many wells.
If you want to risk some wells having more than one clone growing, seed 2
to 5 cells per well. Since some of them might die and there is a gaussian
distribution of cells per well, normally 1 to 3 plates are enough to
isolate 5 high expression clones.
At 22:50 27.04.2004 +0100, Neil Fraser wrote:
>I'm trying to dilution clone a mixed population of transfected HEK293
>cells, but nothing seems to grow!
>When routinely passaged at a 1:10 or 1:20 split they grow OK - albeit
>slowly. I've tried setting up dilutions in 96 well plates from 0.25
>cells/well to 1/well as recommended by others in the lab. to try and get
>single cells/well, but nothing seems to grow. Could it be that they don't
>like being isolated - if so what's the bet way of getting a clonal
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