I need to transfect my cells previous performance my experiment. I will use lipofectamine 2000 but in the protocol it said that I need one plasmid DNA, I read about many things related to plasmid DNA, but I don´t know if I need a specific one or if there is one universal plasmid DNA used for perform transfection with lipofectamine 2000. I will be thank you if anyone can give me some brief explanation or let me know in which paper or book can I get this information?
What protocols are you using? Not sure if I understand your question, but any plasmid DNA of your choice should be fine. What are you trying to transfect into your cells?
http://www.lifetechnologies.com/content/dam/LifeTech/migration/files/cell-culture/pdfs.par.6683.file.dat/lipofectamine%202000%20reagent%20protocol%20v2.0.pdf
I want to check the expression of EGFR (qPCR and Western Blot) and my references papers said they first transfected the cells previews treatment and then check the levels of this protein/gene. They used Lipofectamine 2000 protocole (the protocole came with the reagent and also you can find it on internet). My main point is stimulate the expression of this gene/protein to after that treat the cells and then check the efficacy.
Thank you for help me.
So it sounds like you are looking to transfect a plasmid containing the EGFR gene into your cells then measure its expression after transfection, rather than just measure the expression of any EGFR already in the cells (endogenous expression). If you are following a protocol from a published paper they should provide information on what plasmid they used, otherwise contact the corresponding author and ask. Do you already have a plasmid containing EGFR?
I only found one but I have to order. First I wanted to be sure about if there is not one universal type of plasmid and thanks to you I am more sure about to order that plasmid (containing the EGFR gene). Thank you for you help.
No problem. As far as I know, the construct of the plasmid DNA is irrelevant for lipofectamine and other lipid-based transfections. You may have to troubleshoot the optimal concentration of reagents depending on how well the cells respond in terms of transfection efficiency and cytotoxicity.
any plasmid can be used with lipofectamine protocol as far as i know. so just go ahead with transfection.
You can use any plasmid, the choice is mainly due to compatibility with what you want to express/overexpress in your cells (basically how this plasmid can replicate carry and expres a cloned geene). I always find it cheaper and easier to clone my own construicts instead of ordering them, since you can design your plasmid as you wish (however, companies like invitrogen can design and do any plasmid you wish in case you have not any experience in cloning and you don't want to learn how to do it). I used BAC plasmids that are the most common. You can use either a simple plasmid with the EGFR gene or you can fuse EGFR with a tag (that makes the proteine detectable or visible for immunostaining or any furher purifications). Good luck!
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