I'd like to design a complete protocol for expressing protein in mammalians by using plasmid expression vectors. However, I've never dealt with culture of competent cells, so I'm not familiar with LB, SOC, Agar and all the other are used on this. Does anyone have a protocol about it?
Hello,
I'm not sure to correctly understand what you exactly want to do !? What does LB, SOC, agar or competent cells (all is for bacteria) have to do with protein expression in mammalian cells ? Is it just for your plasmid amplification ?
Yes, it is for plasmid amplification. I have designed my primers including the Kozak sequence, as well as sequences for different restriction enzymes in 5' and 3' respectively. I'm not sure about the next steps. Competent cells transfection, selection of clones and isolation of modified plasmid.
Hi
Based on my experiences, I highly recommend you to use Fugene transfection reagent of Promega, it works in wide range of cells. However before anything you must select your cell line that you want it to express your desired protein. It has some condition that you might consider.
good luck
OK, I better understand where you are in your project !
In my opinion, the best and robust way to clone you first PCR product for amplification in the TOPO technology from Invitrogen/Life Technologies/Thermo scientific (choose the financial version you prefer ;-) ). 2 choices :
1) universal : PCR2.1 topo TA cloning kit. It's an intermediate vector for your expression project in mammalian cells but allows different possibilities of destination vectors (bacterial or mammalian expression vector, tag changes...). Just check that your DNA pol add an Adenosine at the ends of your product.
2) direct : "Your prefered vector" directionnal topo cloning. Faster but more limited options. Here, you need to have specific sequences in your PCR primers to clone directionnaly in one step your PCR product in the vector you have chosen.
All you need to clone is provided in the Topo Kit: competent bacteria, SOC... and the protocol of course !
Just be careful in your primer design and in the choice of your final vector (if you want strong or inducible expression with transient or stable transfection of your mammalian cells -i.e. need of selection gene or not-)
Hope it will help. Good luck !
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning