...clones with pLKO.1 sequencing primer (5’ CAA GGC TGT TAG AGA GAT AAT TGG A 3’). TIP: You may need to adjust...kinase promoter drives expression of puromycin. Puro R Puromycin resistance gene for selection of pLKO...sulfate, and puromycin are located in the “Appendix”. F.2. Determining the Optimal Puromycin Concentration...differently to puromycin selection. Addgene strongly recommends that you determine the optimal puromycin concentration...to fresh puromycin-containing media every other day. f. The minimum concentration of puromycin that results...with finer increments of puromycin to determine a more precise optimal puromycin concentration.) F.3. Protocol...expression of the puromycin resistance gene. f. To select for infected cells, add puromycin to the media at...

...this protocol uses microscopy to determine the percentage of fluorescent cells, these cells can still be...obtaining accurate titers. Check the cells for mycoplasma regularly Do not over- or under-grow your cells... after 20–30 passages. Do not add penicillin/streptomycin to the media. Titer will vary between cell lines...assume one integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple...