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Electroporation/electrotransfection mammalian cells


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#1 losybelle

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Posted 28 June 2011 - 12:33 PM

Hello:

I'm doing electroporation in mammalian cells, I've been reading some protocols, and now i know that the electroporation has to be done in a 1x106 cell/ml concentration, but how many cells should i seed per well (in a 24well plate)? due that some cells will die during the electroporation and other won't be able to atach, i'm afraid that if I use the usual amount for 24well plates, i won't have enough cells atached to my wells. I'm using 0.2cm cuvettes (Biorad) in a gene pulser Xcell
Could any one help me? thanks!!

#2 bob1

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Posted 28 June 2011 - 05:22 PM

Usually when you are doing this sort of thing, the cells are seeded in a bigger area container (e.g. flask or 10 mm plate) and then lifted, electroporated and seeded at whatever density suits your experiment. 10^6 cells should be enough for about 20 wells.

#3 losybelle

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Posted 28 June 2011 - 09:53 PM

Usually when you are doing this sort of thing, the cells are seeded in a bigger area container (e.g. flask or 10 mm plate) and then lifted, electroporated and seeded at whatever density suits your experiment. 10^6 cells should be enough for about 20 wells.


Thanks! i have never done that before so I have a lot of doubts. I thought that the size of the well didnīt matter. So, do you think that it would be better if I try in a 12 wells plate? then 100ul of cells in 1x106 cell/ml should be enough for a well (12 well plate)?
And another question, as I have to do duplicates, can I electroporate 200ul of cells at he same time in a 0.2cm cuvette?

thank you very much!! Your answer was really helpfull!!!

#4 bob1

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Posted 29 June 2011 - 01:03 AM

Just a note to say I didn't mean that you should use bigger wells for seeding out your electroporation, only for growing the cells beforehand, as it is easier and less variable to harvest one flask than it is to harvest wells individually. The size of the well doesn't matter once they are transfected.

The amount of cells you can electroporate at one time will depend on the cuvette and the machine you use. Read the manual, it should have the specific information you need. Having said that, depending on the answers you want, it is often best to do the duplicates at the same time, rather than doing them individually.

#5 losybelle

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Posted 29 June 2011 - 08:08 AM

Just a note to say I didn't mean that you should use bigger wells for seeding out your electroporation, only for growing the cells beforehand, as it is easier and less variable to harvest one flask than it is to harvest wells individually. The size of the well doesn't matter once they are transfected.

The amount of cells you can electroporate at one time will depend on the cuvette and the machine you use. Read the manual, it should have the specific information you need. Having said that, depending on the answers you want, it is often best to do the duplicates at the same time, rather than doing them individually.


Ok! sorry that I misunderstood you when you told me about the harvesting cells, in fact, i'm using 100mm dishes for that. Thank you again for the info. The cuvette can handle till 500ul of cell+medium, so i think i can electroporate duplicates at the same time; i only wanted to be sure about that.
I will try the protocol tomorrow, wish me luck!!

And one more time, thanks!!

#6 losybelle

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Posted 30 June 2011 - 01:07 PM

I'd have another question:

I have to do 3 different electroporations:

-Plasmid 1 + pcDNA
-Plasmid 1 + plasmid 2
-Plasmid 1 + plasmid 3

I'm using 0.2cm cuvettes because i don't have 24 or 96 well electroporation plates. My question is: how should I prepare the samples for the experiment?

I mean, can i do aliquots with the appropriate number of cells and add to them the plasmid and electroporate, one by one? (essemple: aliquot 100ul cell mix/ add plasmids/ electroporate/ seed. Aliquot 100ul cell mix/ add plasmids / electroporate/seed and so on) Or is better to add the plasmids to the different cell groups at the same time and then electroporate them?(essemple: aliquot 3 times 100ul cell mix/ add plasmids to the 3 tubes with cell mix/ then electroporate one mix and seed/ electroporate second mix and seed/ and so on)

Thanks

#7 bob1

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Posted 30 June 2011 - 05:43 PM

It probably doesn't really matter, though in terms of treatment of the cells, it is probably better if you go for the first option so that the cells are not sitting around in transfection mix before electroporation.

#8 losybelle

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Posted 01 July 2011 - 07:57 AM

It probably doesn't really matter, though in terms of treatment of the cells, it is probably better if you go for the first option so that the cells are not sitting around in transfection mix before electroporation.


thanks!!




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