Calcium Phosphate transfection help - cells detaching after media change (Jan/20/2010 )

Hello everyone,

was looking for a little insight to a problem I seem to be having...
so, about a month ago... my calcium phosphate transfection started going wrong - found it weird because I couldn't observe DNA precipitates (either clumpy or fine; nor any sort of trashlike suspension) immediately after adding the mix to cell culture (and also when media changing 8hrs afterward)... so to check, I FACSed my cells after transfecting 293T cells and found that incorporation after 48hrs was only 3-4% (also checked under the microscope and could barely observe green anywhere in my culture)

yep, quite a shock!!

so I had a doctor student do the calcium phosphate transfection simultaneously, using cell cultures that I provided - but both of us using our own reagents... I media changed both cultures afterwards

we both noticed we got very little DNA (GFP) into our cells

we decided to check our reagents and DNA... as an extra control, we transfected an empty vector and a VENUS construct; we also both got new aliquots of Na2HPO4, I also happened to remake fresh CaCl2... the Hepes solution is something we share in the lab and since no one seemed to be having any issues we left that as it was... and so we redid the transfection

results: we both got about 10% GFP in (by FACS) and around 50% Venus in (by FACS too)... under the microscope GFP looked yep, like 10% incorporation, although VENUS looked around 70-80%...

that aside, I seem to be having a new problem which I hadn't experienced before... I transfect my cells, observe particle suspensions which set after 3-4 hours... I media change after 8 hrs, and the next day i look at my culture and around half of them are floating (dead)... I've done this quite a few times but it just keeps on happening

media change doesn't seem to be an issue because I have a control (non-transfected) well that is fine after the media change... I'm guessing it must be one of the reagents (likely CaCl2 - since i made that fresh; I mean, the Na2HPO4 was fresh but my friend didn't have any issues when we transfected simultaneously)... I don't think it's an issue with DNA because I haven't regrown any of my plasmids

any ideas
the cell culture itself does look a little more yellowish than I would like it to (aka. its acidic) right after adding the mix... but that is not something necessarily new and I am wondering if replacing with fresh media... you know, it should be helping right... not the other way around

seriously, any input at all is much appreciated

I did Calcium Phosphate Transfection several times before. It did work well when the system is working. But recently I find a very weird thing about the transfection. So here is the story. I split 6 10-cm dished of plat-E one day before. They are 40-50% confluency on the day of transfection. I transfected 6 different virus plamids exactly the same way. Fine dots formed after 2 hours and everything looks normal. But 24 hours after transfection, when I check them, 2 of the 6 dishes have rounded and floating cells with a lot of dots on the bottom of the plates, while the other 4 dished are just fine.

I have no idea what happened. Are the plamids making that much difference? I extract the plamids using the same maxi prep and from the same batch of LB culture...

Anyone has an explanation?


Helen