Can anyone help me with the amount of DNA, lipofectamine and proper vector control they used for a 96 well plate transfection assay.
Dear Debasish,
The amount of DNA, as well as the ratio DNA:lipofectamine varies depending of the cell type, the vector size, the cell growth rate of your cells... there are many factors that affect the efficiency in the transfection assay, that´s why you should test different conditions to fine tune your transfection protocol.
There are different "Lipofectamines" available. Usually a good start would be to test 1:1 to 1:4 DNA:lipofectamin (but check the supplier guidelines for transfection to do a better approach). They use to present a table where the supplier suggest certain test to reach the best conditions for your experiment (e.g. I´m using Lipofectamine LTX, and the brochure shows a table to test different ratios in a 24well plate http://tools.invitrogen.com/content/sfs/manuals/lipofectamine_LTX_and_PLUS_sample_man.pdf). You could scale down the amount of DNA and lipofectamine to do the trasnfection in a 96w plate).
Are you going to transfect adherent cells? If this is your case, you should take into account that the cells should be in the exponential phase of growing (about 50% to 80% confluent) when you do the transfection. The number of cells to seed varies with the cell type. I´m sorry I cannot be more specific, but if you could give us some details about the cell type I could help you.
I hope this helps,
Aida
A little more details would be great to give usefull advices!
Not knowing what kind of transfection you want to do I can only report the following:
When I want to transfect cellline for the screening of compounds with reportergen constructs I usually transfect the cells in a 10cm dish and plate them to 96well afterwards. This gives very good results!
Dear Debasish,
The amount of DNA, as well as the ratio DNA:lipofectamine varies depending of the cell type, the vector size, the cell growth rate of your cells... there are many factors that affect the efficiency in the transfection assay, that´s why you should test different conditions to fine tune your transfection protocol.
There are different "Lipofectamines" available. Usually a good start would be to test 1:1 to 1:4 DNA:lipofectamin (but check the supplier guidelines for transfection to do a better approach). They use to present a table where the supplier suggest certain test to reach the best conditions for your experiment (e.g. I´m using Lipofectamine LTX, and the brochure shows a table to test different ratios in a 24well plate http://tools.invitrogen.com/content/sfs/manuals/lipofectamine_LTX_and_PLUS_sample_man.pdf). You could scale down the amount of DNA and lipofectamine to do the trasnfection in a 96w plate).
Are you going to transfect adherent cells? If this is your case, you should take into account that the cells should be in the exponential phase of growing (about 50% to 80% confluent) when you do the transfection. The number of cells to seed varies with the cell type. I´m sorry I cannot be more specific, but if you could give us some details about the cell type I could help you.
I hope this helps,
Aida
@ Aida: Thank you for your valuable suggestions.. i am using A549 and MCF7 for these experiments.. but the problem we are facing is with the manufacturer protocol (Lipofectamine 2000, invitrogen) we are not getting much trannsfection efficiency (30-40 %), moreover there is a problem with proper vector control, we have tried with couple of vector controls but they failed to show the results (Giving more cytotoxic effect than p53),
So it would be a great help if you can help me out..
Regards
Debasish
@ Kai: Thank you for your valuable suggestions.. i am using A549 and MCF7 for these experiments.. but the problem we are facing is with the manufacturer protocol (Lipofectamine 2000, invitrogen) we are not getting much trannsfection efficiency (30-40 %), moreover there is a problem with proper vector control, we have tried with couple of vector controls but they failed to show the results (Giving more cytotoxic effect than p53),
So it would be a great help if you can help me out..
Regards
Debasish
If you cannot increase the trasnfection efficiency modifying the Lipof:DNA ratio, i´ll suggest to change to other transfection reagent such us Lipofectamine LTX from invitrogen, as I mentioned before, or TransIT2020 by Mirus (http://www.mirusbio.com/products/transfection/transit_2020_transfection_reagent).
With certain some of the cell lines that i was working with, I´ve compared different transfect reagents and TransIT2020 increase the efficiency 2-3 fold compare to others. I think you can request a free sample from the company.
Otherwise, if the efficiency is still low, Could you change to other plasmid with a selection marker?
I always had problems transfecting my cells in the 96 well format. So I switched to transfecting them in 6 wells and instead of media change after 6 hours of incubation, I seed them in the 96 wells.
@Tanja: Do you mean that you transfect one sample in a single well using 6 wells, then seed them into 3-4 replicates in 96-well plate?
@ Meng-Kiat Kuah Yes exactly. Most times I have the same constructs for several wells. Also the transfection efficiency was not the same when I transfected them individually.
@Tanja: Did you detach the cells from 6-well and then seed them again in 96 cell? Thanks!
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