I am trying to cotrasfect two plasmids into HEK293. One plasmid with cDNA of Clc1 gene (2967 bp) and second plasmid with cDNA of GFP (717 bp). Then I make patch clamp recording of chloride channel. I use GFP for visual detection of transfected cells. But my problem is that many cells (around 50 %), that express GFP don´t have a current. So a suppose, that in these cells is just plasmid with GFP and no plasmid with Clc1 (or Clc1 gene is not expressed? I don´t know). So it is not possible to use GFP like reporter of transfection.
Can anyone know how to cotransfect both plasmids to the cell?
I use Lipofectamine LTX & PLUS Reagent and 35 mm dish (analogous to 6-well), 400 000 cells per dish. My protocol is:
1. Label tube as A: take 150 ul of Optimem and add 5 ul Lipofectamine LTX Reagent.
2. Label tube as B: take 1ug of Clc1 plasmid and add 0,25 ug of GFP plasmid, then add 150 ul of Optimem, then add 3,5 ul PLUS Reagent.
3. Incubate tubes for 5 minutes.
4. After 5 minutes, take everything from tube A and add in tube B.
5. Incubate for 20 minutes.
6. Now add tranfection mix into dish with cells.
7. 4 hours after transfection I remove medium with transfection mix and add fresh medium.
8. 24 hours after transfection I make patch clamp recording.
I tried to change ratio of two plasmids (4:1, 5:1, 1:1), amount of DNA, transfection reagent (Lipofectamine 2000) and nothing helped. I have still many cells that express GFP but no Clc1.
Can anyone help me?
Hi,
I think your problem is that the GFP plasmid is more than 2000 bp smaller than your channel plasmid, so it typically has an advantage in the transfection. Probably a 1:4 (or 1:5) ratio is too small a difference. Try a 1:10 and a 1:20 ratio (remember that in biology a factor of two is almost nothing, there is a lot of tolerance), reducing substantially the amount of GFP plasmid (or increasing the other, depending on how many cells you wish to have transfected... still, 50% is a lot for patching...)
The other thing I would do is to add the plasmids to the Optimem, not vice versa...
Good luck!
The problem with co-transfecting a reporter plasmid is that your reporter+ cells might/might not have taken up your plasmid of interest. It could be that the cells preferentially take up the GFP plasmid (being smaller). You could try the following.
To determine if your Clc1 plasmid is taken up/functional:
1. Do you have a marker like antibiotic resistance in your Clc1 plasmid? If so, add the antibiotic and only the cells that have Clc1 plasmid would survive.
2. If you don't have antibiotic resistance gene in your plasmid, do a western blot/q-PCR for Clc1 on the transfected cells and see if your transfected cells are expressing Clc1.
If the cells have trouble getting transfected with both plasmids and you really want to visualize the transfected cells with GFP, see if there is a way you can sub-clone GFP into your Clc1 plasmid backbone. This is pretty straight forward and will ensure that all your GFP+ cells will express Clc1 gene.
I would ask
1. Is vector size are comparable?
2. Purity or quality of plasmids similar? (will affect transfection and expression too)
3. Promoters same or not in both vectors? (some promoters strong and fast some are not, also half life of the protein)
or
4. simply could be concentration issue (re-check plasmid concentration)
I wonder was that too early to see clc1 effect in all GFP+ cells, and why you choose that ratio 1 : 4 (calculate how many moles of plasmid you are giving to cells 1:1?)
GFP usually very bright and easily seen (if you see carefully some of your GFP+ cells weak some are not so that could be part of reason, meanwhile some other proteins needs more time to be expressed (to reach detectable level)
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