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How long can I wait after seeing HeLa cells till transfection - will it affect my Efficiency (Apr/05/2009 )

How long can I wait after seeing HeLa cells till transfection?
I heard that the optimal is 24 h, but I wander if it will affect my transfection if I will wait 72
I want to seed in 6-well plate before the weekend and to do the lipofectamine transfection in the beginning of the week.

thanks!
:P

-Keshet-

Keshet on Apr 5 2009, 01:03 PM said:

How long can I wait after seeing HeLa cells till transfection?
I heard that the optimal is 24 h, but I wander if it will affect my transfection if I will wait 72
I want to seed in 6-well plate before the weekend and to do the lipofectamine transfection in the beginning of the week.

thanks!
:(


Hi there

Typically you can wait 5/6 hours after transfection before looking at your cells, as with most transfection reagents, maximum transfection is achieved after this time (using Jet Pei, Fugene6 etc). Obviously the longer you leave your cells, the more will be transfected, so it just depends on what you are going to do with them, and how many transfected cells you need.

With regards to longer transfections, 72 hours may be ok, the longer you leave it, the more chance of the cells losing the plasmid, as its not integrated into their DNA, in this case you could set up cells that stably express your gene of interest.

Also, i think you will be fine plating cells on the fri and transfecting on the mon, as long as the cells are not over-confluent, and healthy-looking, maybe try changing the transfection reagent before you do your transfection on the mon!

Hope this helps

Chris

-noakes84-

Keshet on Apr 5 2009, 01:03 PM said:

How long can I wait after seeing HeLa cells till transfection?
I heard that the optimal is 24 h, but I wander if it will affect my transfection if I will wait 72
I want to seed in 6-well plate before the weekend and to do the lipofectamine transfection in the beginning of the week.

thanks!
:(


Hi there

Typically you can wait 5/6 hours after transfection before looking at your cells, as with most transfection reagents, maximum transfection is achieved after this time (using Jet Pei, Fugene6 etc). Obviously the longer you leave your cells, the more will be transfected, so it just depends on what you are going to do with them, and how many transfected cells you need.

With regards to longer transfections, 72 hours may be ok, the longer you leave it, the more chance of the cells losing the plasmid, as its not integrated into their DNA, in this case you could set up cells that stably express your gene of interest.

Also, i think you will be fine plating cells on the fri and transfecting on the mon, as long as the cells are not over-confluent, and healthy-looking, maybe try changing the transfection reagent before you do your transfection on the mon!

Hope this helps

Chris

-noakes84-

I think you are talking about how long after seeding can you transfect?

The answer is that you can transfect while you are seeding, depending a bit on your transfection reagent. Check out Reverse transfection in the Lipofectamine manual. I have also used it successfully with Fugene. Just be aware that altering some genes will affect how well the cells attach so have a look at what you are transfecting and its action in the cell.

-bob1-

bob1 on Apr 6 2009, 01:30 AM said:

The answer is that you can transfect while you are seeding, depending a bit on your transfection reagent. Check out Reverse transfection in the Lipofectamine manual. I have also used it successfully with Fugene. Just be aware that altering some genes will affect how well the cells attach so have a look at what you are transfecting and its action in the cell.


Wow, thanks for this, I never realised you could transfect at the same time as seeding. The lipofectamine manual says you can use this with RNAs, have you ever tried it with plasmids???

P

-Penguin-

bob1 on Apr 5 2009, 05:30 PM said:

I think you are talking about how long after seeding can you transfect?


ya, that what i ment... :( sorry :)

Also, i think you will be fine plating cells on the fri and transfecting on the mon, as long as the cells are not over-confluent, and healthy-looking, maybe try changing the transfection reagent before you do your transfection on the mon!

Hope this helps

Chris



my problem is that we have a holyday here and right after- the weekend.
but i will come during the holyday and the weekend...

thanks for all the tips!

-Keshet-

Penguin on Apr 6 2009, 02:06 AM said:

Wow, thanks for this, I never realised you could transfect at the same time as seeding. The lipofectamine manual says you can use this with RNAs, have you ever tried it with plasmids???

No, I haven't tried it with plasmids, it should still work so long as the protein to be expressed doesn't affect attachment factors.


Keshet on Apr 6 2009, 03:28 AM said:

Also, i think you will be fine plating cells on the fri and transfecting on the mon, as long as the cells are not over-confluent, and healthy-looking, maybe try changing the transfection reagent before you do your transfection on the mon!

Hope this helps

Chris
my problem is that we have a holyday here and right after- the weekend.
but i will come during the holyday and the weekend...

thanks for all the tips!

The one problem with this system is that minor variations in seeding will be amplified by the time you come to transfect, also it makes it much harder to approximate dosages of gene of interest, though this may not be such an issue if your cells are slow growing. Confluency is very important too, if they are too confluent cells often senesce which will alter how much of your gene of interest is expressed.

-bob1-