Hi everyone!
I did a first experience in NIH 3T3 using Invitrogene Neon system:
1x10.6 cell/transfection, eletropo program ; Pulse voltage 1.350V, pulse width ms 20, pulse number 2 (100ul tip). buffer R, buffer E2 in the electro tube.
I had a lot of toxicity (43%) with a poor protein expression (western-blot : don t have a band corresponding to the prot).
I don t have an idea to improve the transfection (I don t have a GFP plasmid yet to visualise the transfection rate... I know, I should have this as it is really usefull).
Any idea to improve my experiment? More cells? another program setting?
Many thanks in advance :).
Alice
I think the best thing is to run the optimization program. In this way you should have the best conditions for your cellular type and you should improve your transfection. In which plate you have seeded 1x10.6 cell? Generally, I use this amount in 100mm plate. Another critical step for the success of transfection is the DNA quality.
G
hi Giuseppe,
thanks a lot for your answer.
As you, I seed my cell after electroporation on a 100mm plate. the quality of my DNA is good (ratio 260/230 between 1.80-2.00).
You're wright there is an optimization program to run before your first assay. Actually, I am in a hurry, and I was wondering if someone could give me a hand :) to save time.
Last week I tried to reduce DNA quantity (10, 5 2,5 ug). It seems that I reduced the tox... (my construction seems to be toxic... not lucky). No huge tox with empty plasmid. Maybe it is a good point if I see tox, that means (maybe) that the cells express the prot (I have to optimize my western blot as well )
In parallel I tried Fugene6. No tox at all (I saw DNA precipitate on dish)...
Next week I will perform western-blot to check the prot expression. I will see (or not).:)
Thanks again a lot.
Do not hesitate for more advice!
Alice
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