I haven't done stables in a while, but these are the steps that I remember. Maybe some others can contribute.

1. Transfect Hek293T at 40–80% confluency with gfp tagged protein vector. This should be done is a dish "do not use a flask"

2. 48hours post-transfection, - Add fresh media containing, Zeocin at a concentration of 50-300 µg/ml for selection in mammalian cells. For 293, it is suggested to use 100ug/ml. You might have to split your cells so that they ~ 25% confluent

3. Every 3-4 days, add fresh media w/ Zeocin

4. After 7 days or longer, you should see single foci throughout the plate.

5. If you are ready to isolate the single foci then (A) remove media from cells (B) circle each individual foci on the bottom side of the cell culture dish (C) place autoclaved plastic selection tubes onto the individual foci (D) add trypsin (E) neutrize trypsin with selection media (F) transfer single or pooled foci to a 35mm dish.

I haven't done stables in a while, but these are the steps that I remember. Maybe some others can contribute.

1. Transfect Hek293T at 40–80% confluency with gfp tagged protein vector. This should be done is a dish "do not use a flask"

2. 48hours post-transfection, - Add fresh media containing, Zeocin at a concentration of 50-300 µg/ml for selection in mammalian cells. For 293, it is suggested to use 100ug/ml. You might have to split your cells so that they ~ 25% confluent

3. Every 3-4 days, add fresh media w/ Zeocin

4. After 7 days or longer, you should see single foci throughout the plate.

5. If you are ready to isolate the single foci then (A) remove media from cells (B) circle each individual foci on the bottom side of the cell culture dish (C) place autoclaved plastic selection tubes onto the individual foci (D) add trypsin (E) neutrize trypsin with selection media (F) transfer single or pooled foci to a 35mm dish.

Thanyou i will try this

Clarification - Add trypsin into selection tube and wait however long you generally do for your hek392T cells to detach.. you don't require very much trypsin, because the tubes are very small.

Thank you again

First you have to determinate the kill curve, that is grown cells (no tranfected cells) in different concentration of your antibiotic. The minimun concentration of your antibiotic that cause complete death after 4-6 days is your concentration to select stable cells.

@Loreto...the cells anyway would die without the resistance, offered from the transfected plasmid or?(i might be totally wrong too)

If you add too little antibiotic - untransfected cells would survive. If you add too much antibiotic - even cells with resistance would die. So, if you add antibiotic to your transfected cells and they survive, unless you have a control with all dead untransfected cells, you won't know for sure that the survivng cells are the ones that are resistant.

If you don't maintain your cells in the antibiotic, they potentially can kick your whole transgene out, but what really bugs me is that sometimes the cells can kick out your transgene, but leave the antiboitic resistance gene... Be careful with this especially after a few passages!

Check this page, it is very helpful

http://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/

Hi Kritan

All the above info is good advice. The only addition I can make is:

- linearise your plasmid prior to transfection (as your plasmid integrates it has to linearise anyway, pre-linearisation does ont affect transfection efficiency however it will give you control over where your plasmid gets cut)

- be careful with Zeocin - it can be difficult to tell if cells are alive or dead (even after a week of Zeocin dead cell ghosts often look alive)

- as you're incorporating a GFP, why not FACS sort out your GFP cells at the end of the first week (unless you want monoclonal cell lines). This had the added benefit that it (somewhat) controls for insertion into a transcriptionally 'dead' part of the genome, or disruption of the transgene cassette during intergration.

Good luck