6 replies to this topic

[shojjahd]

Hi there, please can someone help!

I am trying to do transient co-transfections using HeLa cells and FuGENE 6 reagent. My luciferase reporter construct (in pGL3-basic) is co-transfected with the firefly control reporter driven by sv40. I do these is 96-well plates.
The problem is that I am not really getting any consistent readings even when i do exaclty the same protocol. I have checked DNA purity, for cell infection and all seems fine. I get very dismal numbers for the firefly readings and ok figures for the control renilla. Does anyone do 96-well transfections using HeLa cells that could help? how much DNA should i use/well? Currently i transfect 100ng DNA/well(+12.5ng control vector).

Any advice much appreciated

Edited by shojjahd, 01 March 2005 - 03:43 AM.

[fred_33]

hi

you can see the discussion at

http://www.protocol-online.org/forums/inde...?showtopic=5400

Fred

[shojjahd]

Thank you!

[neuromatt]

If I understand correctly the fact that you are getting a signal from the SV40 driven renilla plasmid and not your pGL3 construct (which I assume contains a promoter that you are studying) means that the transfection is working. The fact that the siganl is poor for pGL3 could mean that the promoter under investigation has weak ativity in your cell line. Have you tried increasing the amount of plasmid?

[shojjahd]

Thank you for the suggestion-

I will try increasing the amount of plasmid DNA and will hopefully see an improvement.

I think what worried me the most was the fact that occasionally, in some of the duplicate wells, i get a good signal for pGL3; whilst in other (identically treated) wells I get very low signals.

Do you think the confuency of the cells the day you passage and seed them have a factor in terms of their overall uptake of the plasmid? Currently i grow them up about 80% confluency and then passage and seed them in 96 wells the day before transfection.

either way, I guess its a case of trial and error.

[shojjahd]

I have tried upping the amount of plasmid by two fold; still hasn't helped.

Is it ALL to do with the exact number of cells in each well? I incubate for 48hrs before reading plates.

[fred_33]

hi
maybe the ratio fugene 6 / dna is wrong? is it a good agent for hela cells?

i have take a look at the lipofectamine protocol. And it seems that for a 96well plate you should use 0.05 to 0.1 µg of DNA
i explain:fo 24 well plate they recommend 0.2 to 0.4 ng
so 96well plate : surface is 4fold less
so if we adjust DNA quantity it is 0.05 to 0.1µg
hence i suppose thez quantity of dna is correct...

regarding your seeding protocol, i think it's ok

Edited by fred_33, 14 March 2005 - 03:30 AM.