Our lab purchased some pLV[shRNA]-LacI:T2A:Puro-U6/2xLacO vectors, the IPTG-inducible knockdown system, from Vector builder a while ago.
Recently, we have 3 students working on 3 different genes (3 shRNA constructs per gene). 2 of the 3 students didn't have any luck in getting the knockdown to work, while the 3rd student is still in the middle of setting up his experiment to test his constructs. Can anyone who has used this system before provide some advice?
They have tested IPTG at 0.25mM, 0.5mM and 1mM at 48h, 72h and 96h. Replenishing IPTG both daily and every other day. I've contacted the company but their advice was very general and we have already done what they have suggested.
Thanks.
Sabine Strehl Yes, the constructs contained a puromycin resistant gene. All cells used for their experiments were selected with puromycin and maintained in puromycin.
Under prolonged exposure to puromycin cells may become resistant, therefore, I am not sure whether it is a smart idea to maintain them on the antibiotic. I would suggest that you design a set of PCR assays to confirm that the desired constructs are indeed intact in your cells.
Sabine Strehl Thanks for your comment. One student managed to get one of the cell lines to work. Will get them to repeat. Might just be something overlooked by them when setting the experiment up (they are very green in this area).
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