10 replies to this topic

[science noob]

My current plasmid of interest was constructed in another lab and doesn't contain the GFP tag, so I wouldn't be able to see transfection efficiency post-transfection.  Would it be possible to co-transfect my HEK293 with another plasmid that has a GFP insert but with another antibiotic resistance? Which system would suit this purposes best - lipofectamine OR electroporation?

What would the transfection efficiency be if I had two plasmids? (one will be fairly huge - 16kb).

Can I safely assume that if the HEK cells were successfully transfected with the GFP plasmid, that those cells would also contain my plasmid of interest (16kb)?

[prabhubct]

Whether you origin of replication is same or not? Same Replication plasmids are incompatible. You need to use both Selection marker's simultaneously for selecting cells containing both plasmids (with different Ori.)

[bob1]

You can't assume that the transfection efficiencies will be the same for both plasmids - you can do immuno to assess the rates for each.  The bigger the plasmid, the poorer the transfection usually.  I would go for electroporation for large plasmids.

You also can't assume that cells that express GFP will also have your plasmid in them, though this will often be the case.  You would need the same selection marker on both plasmids to make co-transfection more likely.

[prabhubct]

bob1, on 20 September 2012 - 01:26 AM, said:

You would need the same selection marker on both plasmids to make co-transfection more likely.

Why same selection markers needed? why not different?

[bob1]

prabhubct, on 20 September 2012 - 03:52 AM, said:

bob1, on 20 September 2012 - 01:26 AM, said:

You would need the same selection marker on both plasmids to make co-transfection more likely.

Why same selection markers needed? why not different?

Because if you have different selection markers you will be selecting with 2 different selection agents at the same time, or you will only be selecting for one of the plasmids...

[leelee]

I'm confused too, bob1.

If you have the same selectable marker on both plasmids, then how will you know that both have been successfully transfected and not just one or the other? As the cell will only need one to survive. And wouldn't that fact also encourage the cell to lose one or other over time? As it only really requires one?

Or am I missing something?

[prabhubct]

bob1, on 20 September 2012 - 01:39 PM, said:

prabhubct, on 20 September 2012 - 03:52 AM, said:

bob1, on 20 September 2012 - 01:26 AM, said:

You would need the same selection marker on both plasmids to make co-transfection more likely.

Why same selection markers needed? why not different?

or you will only be selecting for one of the plasmids...

Can you please elaborate more. thanks.

[bob1]

@leelee: correct you can't tell, but selecting for only one will guarantee that the other is lost.  However, you can't tell if you have co-transfected anyway, unless  you have a means of assaying for both plasmid's products.

@prabhubct:  If you only select for one plasmid the other will be treated by the cells in a simiar manner to a transient transfection, and will be lost after a few days.  Double selection using different agents can be done, but it is very hard on the cells and unless carefully optimized results in a lower colony formation rate than doing the selection sequentially.

"more likely" was a poor choice of words by me, what I should have said is that cells that survive will have either one of the plasmids or both, but using a plasmid with a different selection marker will result in that plasmid being lost unless it is also selected for.

[Trof]

AFAIK would be better to redesign the plasmid, unless the GFP is on the same plasmid, it would be useless for checking transformation efficiency if it has much higher transformation efficiency than your target.

[Curtis]

Trof, on 21 September 2012 - 08:36 AM, said:

AFAIK would be better to redesign the plasmid, unless the GFP is on the same plasmid, it would be useless for checking transformation efficiency if it has much higher transformation efficiency than your target.

transformation?

I thought 16 kb is already in the range. I have seen people transfect >18 kb plasmids with Fugene.

[Trof]

Transfection, sorry.

The limiting size is not the problem here, the question is not whether it's possible to transfect with two plasmids, the question is, if he can transfect two separate plasmids, one with obviously different efficiency AND use the second plasmid to calculate the efficiency of the first.

It would be like measuring how much salt can dissolve in an amount of water by measuring how much sugar dissolves there.