Hi Daniel,

I usually start selection after appr. 18-24h post transfection. Since I am not using synchronized cultures I guess this would be after the first division.

This worked well for Blasticidin, Hygromycin and G418. To get your selection to work fast and if you are interested in mixed clones, splitting the cell more often than usual helps pretty well.

I have to admit, that I never thought about adding the selctioning drug earlier, since the cell proteinbiosynthesis could be blocked even before the proteins providing resistance are translated.

Earlier addition of selectioning drug would most probably enhance its effectiveness.

Best,

Kai

Hello Daniel,

I typically transfect and after 8 hours of transfection (in 6 well plate), I switch the media to back to normal growth media. After 24 hours I expand the cells into a T75 flask. After another 24 hours, I begin selection. This gives my cells plenty of time to recover from transfection before hitting them with Zeocin and has worked quite well. This is for mixed pool selection.

For clonal line development, I transfect as described above but instead of diluting the transfected cells into a T75, I dilute them into 20mL and distribute 50uL aliquots into 96 well plates. After 24 hours in the 96 well plates, the media is replaced with selective media (+Zeocin).

So, in both cases, I've had success with adding selection after the first cell split. Hope this helps.

Jason

I generally tend to generate polyclonal cell lines myself - and have primarily used vectors with blasticidin, puromycin and G418 (of these, puro tends to be the most effective killer). Usually, I transfect cells at a reasonable conflurency in a 60mm dish. 24h later, I split the cells from a 60mm dish into a T75 - putting them directly into selective media (+ predetermined effective killing concentration of antibiotic). I have found this typically works better than adding selective media to cells for a day or two before splitting. I suspect this is because some resistance can be transferred between cells on contact.

On a related note, I always make sure that i have one untransfected dish that I treat the same way (splitting into selective media) to ensure that the antibiotic is working effectively and there is an effective kill of untransfected cells.

I usually split the cells (1:40, or 1:80) 24 hours after transfection, then directly add the drugs. The dilution ratio depends on the transfection efficiency and cell doubling time. It's better to use the ratio which can give single isolated colonies on the plates. Change the medium every 2 days. Puromycin is the best so far.

After the first division, so we usually wait at least 24hrs.

Thanks everyone. I usually add drugs just before the first division. It has worked in most cases but recently l seem to have a few problems. I will try after 24 h just to be sure the problem is not with the transfection but the gene l am knocking out. Thanks again