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transfection of MCF7 and MDAMB231


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#1 vetticus3

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Posted 16 March 2005 - 07:44 PM

Hi,
I'm going to be doing some transfections of MCF-7 and MDA-MB-231 cells, with a plasmid containg CAT, and my gene of interest. We have lipofectamine 2000, and I'm just going through the protocol that is suggested by said manufacturer...
I will be using a 12 well plate, so, according to protocol, I should use 1.6ug DNA diluted in 50uL .... blah blah blah.... but in some papers i've been reading, they've used in a 24 well plate 12 ug of CAT plasmid. Help? This is a huge difference, and i'm not sure if a, this is a typo in the paper (maybe 1.2ug?).
Also, according to manufacturer, transfection should take 24-48 hours.... other papers say 6 hours. Which is best a little time, or a lot?
And another thing, someone suggested that the cells should have a small treatment in glycerol after the transfection medium is removed, for 10 minutes or so. I've never heard of this before, and can't find anything relating to this? Suggestions really appreciated.

:P help me please, limited time frame to do this work in. help me, i've fallen over and can't get up.

Thanks,
vetticus


/ how do you tell if transfection has taken place? facs or something? have no idea on this.

Edited by vetticus3, 16 March 2005 - 07:50 PM.


#2 fred_33

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Posted 17 March 2005 - 02:08 AM

hi
for transfection, manuals talk about 24-48h. This is time for cells to be ok with all the procedure and the minimum time to analyze effects on cells (lysate and/or morphology/doubling time...)
When parpers said it takes 6hours : they probably mean that you let your transfection mxture 6h on the cells and after you complete with a complete medium containing twice the serum concentration (to get a final 1x serum medium) or replace the mixture by fresh complete medium.
In the paper you read, do they use lipofectamine 2000? I think that for improve the transfection efficiency, they increase the dna quantity. I use 12g of plasmid for a 15cm plate!!! Honnestly, i think the comma misses and it's 1.2g.
So see for your experiments if you can increase the dna quantity and if it worse it...
the glycerol treatment is particulary advised for cells which should be handled with care. I've never done it due to the fact i use resistant cells (293, hela...) but it may decrease the amount of cells dying after the transfection.

how do you tell if transfection has taken place? facs or something?

The single way i know in order to check this is to make a co-transfection with a plasmid containg a reporter gene (GFP or DsRED protein) and analyse by fluorescence microscopy or FACS (better).


I hope i've explain you your questions.... And sorry for this so long reply...

Fred

#3 vetticus3

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Posted 17 March 2005 - 08:52 PM

thankyou!!

i thought that 12ug was a bit too much.
we are going to be using luciferase to monitor things.
would antibiotics have an effect on transfection?
this is starting to worry me because, in our lab (not me, i've never done this before) the transfection rate is really (*really*, less than 1%) bad, but we're using a virus, and well it's just badness. but this is a cat, and well things have to work.

anyway, thanks heaps, you rock!

vetticus

#4 fred_33

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Posted 18 March 2005 - 01:18 AM

hi
do not use antiiotics during transfection .
usually i wait 24h to re add antibiotics in the medium. And i never got contaminations.
Regarding your transfection rate, it's quite surprising :D are you tranfecting bunkers? well i think you first have to set up ytour transfection conditions before wasting lot of plasmid (except if you want to make stable cell lines but it gonna take a long time (1month?)) Use your luciferase containing-vector and try different amounts of transfection agent for a defined quantity of dna, and on the other hand try different quantities of dna for two defined transfection agent quantitites. And pick up the best one (s).

fred

you can see the discussion below (it's quite long sorry)
http://www.protocol-...?showtopic=5580

for adding selection in the media :
http://www.protocol-...?showtopic=5555

Edited by fred_33, 18 March 2005 - 01:25 AM.





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