How long for puromycin selection for PX459 in Hek293FT cells?

[Jerry Xu]

I've transfected PX459 into HEK293FT cells, after 24 hours transfection, I add 2ug/ml puromycin for selection. 

After 24 hrs selection, almost all control cells died, which is good. For the sgRNA-transfected cells, it's a mix of healthy cells (supposed contain plasmid) and those floating ones supposed to have died.

But I have several questions:

1. How long should I keep cells under puromycin selection for HEK293? Zhang lab protocol suggest 48-72 hrs. But I read others saying at least 72 hrs. And what about other cell types? I guess all around 2-3 days?

2. Since it's a mix of healthy and dying cells, should I change medium to get rid of the dying cells in case dying cells release nasty stuff?

3. When harvesting cells, I simply need to remove medium and wash PBS and trypsinize cells? But HEK293FT is so easy to get detached so usually I won't wash with PBS; but here for selection I must get rid of the dead cells. .......I have to wash 

4. In addition to harvesting cells for genotyping, I guess I can passage the genome-edited cell for maintanance. For passage I just use normal medium without puromycin? 

Sorry for bugging with so many stupid questions.thx

[Rocamar]

Hi Jerry 

Didier fesquet 

Le 12 oct. 2015 à 05:49, Jerry Xu <gerryb...@gmail.com> a écrit :

I've transfected PX459 into HEK293FT cells, after 24 hours transfection, I add 2ug/ml puromycin for selection. 

Why are you using 293t cells...if you are using thèse for sgguide validation, i would use héla or other more friendly cell lines..simply check the ploidy at the targeted loci.

After 24 hrs selection, almost all control cells died, which is good. For the sgRNA-transfected cells, it's a mix of healthy cells (supposed contain plasmid) and those floating ones supposed to have died.

But I have several questions:

1. How long should I keep cells under puromycin selection for HEK293? Zhang lab protocol suggest 48-72 hrs. But I read others saying at least 72 hrs. And what about other cell types? I guess all around 2-3 days?

Keep the selection as long as you observe living cells in the non transfected population, then remove the puromycine, I usually change the medium everyday during the puro selection.

2. Since it's a mix of healthy and dying cells, should I change medium to get rid of the dying cells in case dying cells release nasty stuff?

Yes 

3. When harvesting cells, I simply need to remove medium and wash PBS and trypsinize cells? But HEK293FT is so easy to get detached so usually I won't wash with PBS; but here for selection I must get rid of the dead cells. .......I have to wash 

This is why I suggest to use an other cells lines.

4. In addition to harvesting cells for genotyping, I guess I can passage the genome-edited cell for maintanance. For passage I just use normal medium without puromycin? 

Yes , do not use puromycine...

Sorry for bugging with so many stupid questions.thx

There is no stupid question..we are here to learn

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[Jerry Xu]

Thanks again.

Very good point that Hela may be better choice. This is my first try for CRISPR so just follow feng zhang protocol....
Also HEK293FT is very easy for transfection. Hopefully to get better transfection efficiency