Hi Mason,

The blobs of strong GFP signal are not expressing cells.  They are usually some dust particles or other random detritus in the well or on the bottom of the well/plate/slide.  The fact that you see them when you change the focus implies that it is probably junk on the bottom or top of the plate.  They can also be fragments of dead cells, as dead cells usually auto-fluoresce in the green channel.  What cells are you trying to transfect?  

I agree with jonathan in what you are seeing is some detritus or fragments of dead cells in another plane of the focus.

In my expierence, to observe a better transfection rate you should wait at least 48-72 h, depending on the doubling time of your cells.

Hi there,

Have you checked the transfection efficiency?

I was trying to check the transfection efficiency with the GFP. It appears that the transfection didn't work.

I'm using calcium phosphate... I'm not sure what i did wrong.

Thanks for the responses! I'll try again tomorrow.

Yeah, you should always use a non-transfected control.  This allows you to identify fluorescent structures that aren't attributable to your GFP. 

Calcium Phosphate is finicky.  You can get it to be effective, but only with a lot of multivariate tweaking (concentrations, times, mixing speeds...).  It's never exactly as advertised. 

Lipid transfection is a lot more user friendly, and less sensitive to seemingly random fluctuations.  But also more expensive.  Maybe get a little bit of transfection lipid somewhere, see what that does. 

Hey John,

I did try an untransfected well (at least a well w/o GFP transfection). No green blobs. I had forgotten about that, thanks for reminding me.

You know, I would truly love to be using lipofectamine, but our boss has decided to phase it out. I guess that makes some sense, as some of the results could be attributable to lipid signaling.

Good discussion everyone, thanks for the help.

-MS

With calcium phosphate method if you are able to see after 24h its great. Even by lipofection method you will be able to see after 48-72h. t is also dependant on what type of cell line you are using. There are hard to transfect cell lines. For them electroporation or nucleofection method will work. The quantity of GFP construct and no. of cells you are using also matters.

Thanks for the advice!

I agree with most comments. CaPO transfection can be tricky and lipofection is more friendly user. Depending on the cells you are using I can send you a free sample of DreamFect Gold for your transfection

http://www.ozbiosciences.com/transfection-dna/19-dreamfect-gold-transfection-reagent.html

Let me know at [email protected]

Good luck

Best Regards