Hi all,
I am trying to establish stable drosophila embryonic line S2R+ and planning to use hygromycin (have pCoHygro plasmid for co-transfection)as a selection drug for the transfected cells. I started selection and encountered almost "contaminant" looking particles in the culture after i added hygromycin. So, i went down to check if my media or hygromycin in our lab is contaminated and surprising saw similar particles in the Schneider's full media supplemented with hygromycin alone immediately after adding the drug. I have double checked media alone (was clear, no suspicious particles) and media with hygromycin from other lab. Also let other person do it for me and got similar precipitates(ppt) with media+ hygromycin. However DMEM with hygromycin doesnt make those ppts.
Has anyone else encountered similar problem? At the moment, I only have pCoHygro plasmid for co-transfection. So it is almost the only antibiotic that i could use for my stable cells.
There's something odd regarding hydromycin in that even manufacturers admit there can be impurities:
http://www.hygromycin-evopure.com/Hygromycin.html
To be sure it's pure, I would run the hygromycin through IR spectrometry. Also, does the medium contain calcium chloride and/or sodium bicarbonate? I imagine calcium may precipitate out with the hygromycin, but it's most likely a contaminant on the hygromycin.
Hey Tomas, thank you for the reply. Schneider's media does contain CaCl2 and other inorganic salts. I would rather change to a newer lot of hygromycin or try some other drugs if i can find other vectors with a different drug selection.
Yeah, I would agree, hygromycin has this tendency to precipitate out (particularly if it has impurities) and the literature tends to be all over the place regarding cause, if you can try a newer batch of hygromycin. If other sources of hygromycin don't work, maybe pCoBlast with Blasticidin S? To be perfectly honest, plasmids are not my expertize but when I can I prefer to synthetize my own chemicals and just looking at Blasticidin S it looks much more stable in solution since my suspicion with hygromycin is that it forms chelates with calcium or other cations and since it's a glycoside it has a high tendency to hydrolyze, but this are just conjectures, I'm sure there are people who understand the topic more deeply. Best regards.
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