Basic steps of stable transfecton? - (Mar/07/2006 )
Hi,
I can´t find a site were anybody explanes the basics of stable transfection in a practical manner. Either it is too basic or to comlicated or the protocol site is no longer available as I saw in protocol-online.org
If I have a tumor cell line and I want to stable transfect it with GFP with G418 as a selection marker. How can I do this and especially what do I need except the cell line itself, were can I get this from. Do I need special technology. In our lab we only make cell culture an fluorescence-microscopy no PCR...
Can I buy the DNA?? associated with the selection marker?? In what case do I need a plasmid-DNA?
Thank´s a lot for help!
AKdS
You can buy the plasmid from clontech (www.clontech.com), invitrogen (www.invitrogen.com), stratagene has some (www.stratagene.com), as has promega (www.promega.com) and probably severall other companies. Most of the GFP's are variants of the original GFP from aquoria victoria, but some are from different species (those from clontech). You better search for the specific applications of your work, to find out which GFP suits you best. It's always plasmid DNA you need, the plasmid contains a bacterial origin of replication, a bacterial selection marker (like gene coding for ampicillin or kanamycin resistance) (these two make sure you can grow bacteria that will contain the plasmid and afterwards you can purify the plasmid), a strong eukaryotic promotor to drive expression of GFP, and a eukaryotic selection marker (f.i. for G418 selection it's the neo-gene if I'm not mistaken).
Then you have to do the transfection, there's a lot of reagents available to do so (search on the mentioned websites for "transfection reagents" or just "transfection" and then look for the reagent that suits you best, meaning high transfection efficiency in your cell line, or cell lines comparable to yours and a low toxicity in this cell line).
Then you just follow the protocol that comes with your transfection reagent and after some time (look in http://www.protocol-online.org/forums/index.php?showforum=7 for when to start your G418 selection, you better do a dose-response curve on non-transfected cells to know which dose of G418 you need) you start selection.
It's always plasmid DNA you need, the plasmid contains a bacterial origin of replication, a bacterial selection marker (like gene coding for ampicillin or kanamycin resistance) (these two make sure you can grow bacteria that will contain the plasmid and afterwards you can purify the plasmid), a strong eukaryotic promotor to drive expression of GFP, and a eukaryotic selection marker (f.i. for G418 selection it's the neo-gene if I'm not mistaken).
Thank you "vairus" for your rapid reply!!
Did I understand right:
I have to buy the plasmid first
I have to grow bacteria with the plasmid????
I have to isolate the plasmid????
I have to do the transfection by protocol of the kit
I have to grow my probably transfected cells
I have to select them by the selection marker
I wish all of you the best day today!!
AKdS
Is this your homework?
Hey vairus,
did I ask something wrong?
No, this is not my homework and I am not a student. I am working in a very small groop of mostly postgraduated students and no one ever did transfecton before. Our contact to other groops does not include one who already did this work. So I found this forum and thought someone could explane it to me. I only want the confirmation of the steps I asked, because if we have to grow bacteria I think we cannot do the transfection.
Of course I am also searching in the internet, but many of the informations are incomplete and start at a certain point of work or end before the whole work is done. Some explain it very detailed. I first have to decide whether we could do this work at all with our equipment. We don´t have the budget to buy instruments. (Oh my god what a poor country we are going to be.)
So if you decide to help me, just tell me the basic steps.
I thank you very much!
AKdS
Owkay, It's just that there's a lot of people who ask their homework questions here, and mostly people who will do transfections have a little more knowledge about basic molecular biology.
You will have to grow the bacteria to purify large quantities of plasmid. Or you can buy large quantities of the plasmids (for instance if you buy from clontech you will get approximately 20 µg of plasmid DNA which will be enough for some transfections, but if you need to repeat your experiments... you'd have to buy it again (thus growing your own bacteria with plasmids will be cheaper in the long run).
For growing the bacteria you will first of all need competent cells (available from a lot of companies in the first post), a waterbath (to get the plasmid into the bacteria) a shaking incubator (for recovery of bacteria after heath shock and later on to grown bacteria) and a regular incubator (to plate the transformed bacteria) together with both liquid and solid LB medium (and preferably a -80 freezer to store transformed bacteria for longer periods).
Maybe you can contact some companies that will grow you large amounts of plasmid DNA at a reasonable price (for instance qiagen does this: http://www1.qiagen.com/Products/Plasmid/Pl...ce/Default.aspx but they probably only do this for very large quantities).