Hi,

1) the antibiotic concentration used for the selection is important to kill only negative cells. Make sure you are using the optimum concentration by performing the concentration dependent cell death observation for 4-14days depending on cell line and antibiotic (puro/G418). Better use two different concentrations : low to select positive population and higher to kill escaped negative cell population.

2) Trasduction might be weak, so try to increase the viral titer or incubate for longer time with virus with intermittent media changes.

All the best.

Hi Shekhar,

Thanks for your clarification. Continuation of previous section, I have transfected the RetroPack PT-67 packaging cell line and I have picked the colony and gave few more days antibiotics treatment.

How long I could use this cell line for generating the virus, when I should collect the virus, and how I should collect the virus from the supernatent?

Thanks.

Antibiotic concentration may be high. You can use after dose check.

For transduction use more

You can try in this way please.