11 replies to this topic

[cm13]

I have a GFP tagged plasmid that i tried to transfect into cells before.
The last few times i did it, it turned out fine.
It needed some optimisation, but eventually i got to find out what the appropriate concentrations were.

I later repeated the same concentrations on cells, and it didnt transfect. The control did though.
I'm not sure what could have gone wrong.

My cells were at passage 11, which is usually the last usable for the type im using (Human aortic endothelial cells).
I thought it could be that the cells were just unhealthy, However, the control (GFP on its own) transfected in.
Is it possible that the cells were just unhelathy for transfecting in a larger plasmid?

Another thing that came to mind was with the plasmid itself. It was eluted in water and stored at -20C, but at one point there was a problem with freezing. I noticed at one point when taking things out of the freezer that the plasmid was not frozen. Is it possible that it could have degraded?

I dont know what else could have gone wrong. Any ideas?

[Curtis]

The same problem happened to me last month and I decided to extract new plasmid from my transformed bacteria.

Although plasmid and DNA are very stable at any temperature, they might also degrade. I'm saying this because I ran my pFLAG on gel and it gave me a big smear. I don't know what happened really. we also had problem with our freezer as it was overloaded and couldn't function well.

my suggestion, just prepare new plasmid from stock to be on the safe side.

[Inmost sun]

what about confluency of cells? Did you transfect at different stages of confluency? one may not succeed with transfection at high or full confluency...

[cm13]

Inmost sun, on 31 August 2010 - 11:43 PM, said:

what about confluency of cells? Did you transfect at different stages of confluency? one may not succeed with transfection at high or full confluency...

Really?

I transfected the cells at 100% confluency when i did that experiment.

Since then, I checked the plasmid on a spectrophotometer. It still seems fine.

I repeated the experiment with cells that were at about 80-90% confluency, and a passage lower.
(The other cells were at passage 11, which is the last one that is workable)
I also trypsinised them with a lower concentration of trypsin (0.5x as opposed to the 1x used last time.)


They didnt transfect again. should i maybe try a lower confluency? I was thinking of working with cells that are only 50% confluent, as if theyre still dividing, they might be more willing to take in a plasmid.

Any other suggestions of what else i should try?

[HomeBrew]

cm13, on 02 September 2010 - 01:27 AM, said:

Since then, I checked the plasmid on a spectrophotometer. It still seems fine.

This won't tell you anything about the condition of the plasmid -- you need to run a sample on a gel to check for degradation.

[cm13]

How will i be able to tell if it is degraded?

[laurequillo]

cm13, on 02 September 2010 - 03:22 AM, said:

How will i be able to tell if it is degraded?

running in a gel as suggested.
For transfection 100% confluency is no the best scenario! I would try 50-70% confluency.

[cm13]

laurequillo, on 02 September 2010 - 03:38 AM, said:

cm13, on 02 September 2010 - 03:22 AM, said:

How will i be able to tell if it is degraded?

running in a gel as suggested.
For transfection 100% confluency is no the best scenario! I would try 50-70% confluency.

I meant what sort of bands should i expect?
Just one?

Im going to try the transfection later.
But first im running a gel.

[laurequillo]

cm13, on 02 September 2010 - 03:41 AM, said:

laurequillo, on 02 September 2010 - 03:38 AM, said:

cm13, on 02 September 2010 - 03:22 AM, said:

How will i be able to tell if it is degraded?

running in a gel as suggested.
For transfection 100% confluency is no the best scenario! I would try 50-70% confluency.

I meant what sort of bands should i expect?
Just one?

Im going to try the transfection later.
But first im running a gel.

If your plasmid is degraded you will get a smear. If not you will get 3 main bands (supercoiled forms)

[cm13]

cm13, on 02 September 2010 - 03:41 AM, said:

laurequillo, on 02 September 2010 - 03:38 AM, said:

cm13, on 02 September 2010 - 03:22 AM, said:

How will i be able to tell if it is degraded?

running in a gel as suggested.
For transfection 100% confluency is no the best scenario! I would try 50-70% confluency.

I meant what sort of bands should i expect?
Just one?

Im going to try the transfection later.
But first im running a gel.

Ok, ive run the plasmid (and a second plasmid that ive to transfect later) on a gel.
What do people reckon?
Too smeared?

 90k plasmid.jpg   38.11K   22 downloads

[cm13]

Bump.
After leaving this for a while, i came back to it. I used cells at a lower passage, and tried different transfection methods, and optimatisations.
As it stands, i have my control GFP transfecting in fine into the cells.
And the cells look reasonably healthy afterwards.

But i'm not getting anythign with my target gfp.
I do see some small lumps of green fluorescence outside of the cells however.
What are these?
Is it a case that the plasmid is too big to go in, or what could be wrong here?

[KevinK]

What is the your target GFP?  Is it a protein fusion betwen GFP and your protein of interst?  Is so, that protein willbe subject to localization and/or degradation depending on the fusion partner.  The other possibility is that there is an issue in the genetic construct.

Hope this is helpful.

Kevin