Hello Kristen Navarro

Before your actual experiment, you will have to first perform a pilot experiment namely, "an antibiotic kill curve". This experiment will help you to determine the optimal concentration of antibiotic that you will be using to select the positive clones.

An antibiotic kill curve is a dose response experiment in which mammalian cells are subjected to increasing amounts of selection antibiotic to determine the minimum concentration of an antibiotic that can kill all the cells in a specific period of time (say over the course of 2 to 7 days). This is a crucial experiment which you should perform before using a selection antibiotic to kill non-transduced cells and select the positive clones.

For puromycin, the optimal concentration is the lowest concentration that kills 100% of non-transduced cells and shows maximal survival of transduced cells in 48-72 hours. Please note that the optimal concentration will be cell type dependent.

For the puromycin kill curve experiment, you may refer to the protocol given below.

https://goldbio.com/documents/1094/Titration+Kill+Curve+Protocol.pdf

Then later you may perform your experiment using the optimal concentration of puromycin which you have determined from the above pilot experiment to select the positive clones.

As far as I know, ampicillin might not be strong enough to allow efficient selection of transduced mammalian cells. You need to confirm this. It can only be used for bacterial selection. Mammalian cells selection requires use of puromycin, geneticin, zeocin, hygromycin, or blasticidin.

Good Luck!

Malcolm Nobre - In the table, is the amount given in the "Volume of Stock Solution Added (μl)" tab for 10 mL of media?

I agree with Malcolm Nobre but I also wanted to add that puromycin is a very potent antibiotic which has a working concentration range of around 0.5 - 10ug/mL for mammalian cells. Cell lines differ as to how sensitive they are to antibiotics. Personally, I think that if all cells die within 48-72 hours the concentration of puromycin might be a bit too high (the idea is to find the lowest concentration that kills all cells; usually, antibiotic titration protocols recommend to treat cells for 1-2 weeks).

Ampicillin is most probably for bacterial selection (i.e. when you are propagating the vector in E. coli). If your vector is available on Addgene, you'll be able to confirm this (they usually specify what antibiotics to use for bacterial selection ('Bacterial resistance') but also for mamalian selection ('Selectable markers').

I believe that the volumes given in the table are not for 10mL of medium, but for 1mL (if you see Step 2, it says 'add 0.5mL of cell suspension to 24-well plate containing 500uL of medium + antibiotic). Also, I wouldn't really pay attention to what they say on Step 5 about recommended concentrations for certain cell lines - the best way is always to make your own antibiotic titration experiment by testing the whole range of working concentrations for each antibiotic.

1 ug/mL puromycin (make a 1000x stock of 1 mg/mL in H2O and filter sterilize) works great for the *vast* majority of cell lines. Just try that and see if it works. You should see significant numbers of floaters within 24 h. Rarely, a higher concentration is needed, probably due to high endogenous activity of CYP3a4 which processes puromycin.

Ampicillin is not used for eukaryotic selection. The gene is in the original plasmid packaging vector solely for propagating in E. coli and is not normally incorporated in the lentivirus particle.