The culture looks fine during maintenance but after transfection, precipitation forms immediately after I adding the antibiotics (hygromycin b) for positive cells selection. So I tested the fresh cells at the suggested cell numbers (6x10^5)for transfection, the precipitation also developed immediately after I adding in the pen/strep or zeocin (CV-1 resistant to this antibiotic). Does anyone have an idea what was going wrong?
Thank you!
Do you have more information regarding your transfection procedure? What reagent? What are you diluting your DNA in?
Are your cells actually precipitating off or are you observing precipitation of something and your cells are still attached?
What are your controls? Have you added just transfection reagent only to your cells followed by antibiotics to see if this still happens or just DNA and no transfection reagent?
Some methods suggest antibiotic-free transfection and then at next media change you introduce the antibiotics. Something to do with interference between reagent:DNA complex. You could try this?
Some lipid-based transfection reagents may produce some precipitation if there is excess EDTA in media/DNA diluent - you can try making your DNA mix up in purified water if this is the case?
Hope it helps...
I use Lipofectamine 2000 for transfection, diluting DNA in Opti-MEM.
The cell (6 x10^5) was prior incubated for a day before transfection without antibiotics. Also, the selection antibiotic (hygromycin) was added only after 48 hours of transfection.
My positive control was a plasmid expressing GFP, the negative control was untransfected cell (I didn't add transfection reagent to save the reagent) but both controls showed precipitation so I suggest the problem is from the cells. Plus, the tests that I have done with Pen/Strep and Zeocin at 6x10^5 number of cells are those after trypsinised/unattached cells (but already washed with PBS and diluted in DMEM), both showed precipitation.
I extract plasmid DNA using midiprep from Qiagen and resuspended using molecular grade water.
Hmm, I've not used CV-1 cells before, but I've worked with fibroblast cells previously. I've not see such a phenomenon when I've used pen/strep or zeocin before...
You could try looking at what the precipitate is - but this is quite time consuming and laborious. Does the precipitation occur after you perform a media change with fresh medium containing the antibiotics?
It could just be an artefact that is produced as a result of using the antibiotics - I'm guessing you have no issues with expression in your transfected cells?
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