I am currently given quite a lot of vectors to insert my gene of interest and I have no idea which one to choose. All of them have restriction sites while some have introns and all of them have different promoters ranging from SV40 promoters to T7 promoter. Which vector should I choose if I am working on a mammlian cell and why?
2006-11-09 01:47:10 · 2 answers · asked by PIPI B 4 in Science & Mathematics ➔ Biology
The main question is: do you want to make transient or stable transfection. If you want to do stable transfections, then you will need a selective marker such as resistance to G418.
Some vectors also code for a fluorescent protein such as GFP or add a tail to your protein such as a His Tag. This helps for the visualization of your protein or the purification but beware of potentially inactivating additions.
You will always have a polycloning site so what you want to find is a set of enzymes that you have in the lab in a good amount and work with them. Find the vectors that have their restriction site for the insert.
As for the promoter of use, ask around in the lab for what works the best with your cell line. It's not such a big choice.
2006-11-09 04:31:00 · answer #1 · answered by TonySti 2 · 0⤊ 0⤋
What are you trying to accomplish? I would use a promoter for sure maybe a tata box. This would help you in protein detection to help select out the cells where tranfection actually occured. I would also use an antibody resistance gene so that you can grow it up on a plate with the antibiotic on it to help for selection as well.
2006-11-09 01:56:54 · answer #2 · answered by ewtaylor2001 5 · 0⤊ 0⤋