Lipofectamin 2000 and Large Plasmid Transfection Optimization - (Nov/16/2012 )
Hi All,
First time posting here, so sorry if I make any mistakes.
I am trying to transfect Bovine Pulmonary Artery Endothelial Cells (BPAEC) and I've been having some issues. My plasmid is about 7744bp and my cells die with 16 hours after transfection with Lipofectamine. Here's what I do for a 35mm dish:
1. Plate cells until 90% confluency
2. 4 ug of DNA in 250 uL of MEM (NO FBS or Antibiotics) and let it sit at RT for 5 min; meanwhile I also mix 10 ul of L2K to 250 ul of MEM (ALSO no FBS or Antibiotics) and let it sit @ RT for 5min
3. After 5 min are up, I add the L2K to the DNA and let it sit for 25 min
4. After 25 min are up, I add to the dish of cells (which contains medium with 10% FBS and NO ANTIBIOTICS)
5. 6 hours later I change my media and let it chill O/N (after 6 hours confluency is about 60% already)
6. Check overnight, and can see the GFP tag but the confluency is about 20%
My positive control is C3-GFP and I get 70%-80% confluency O/N with 60% efficency of transfection.
Don't know what's going on though. Is my plasmid too large for L2K?
Your plasmid isn't the problem in terms of size, though the bigger it is, the harder it is to transfect; 8Kbp is not all that big. L2K is quite toxic by itself so it may be helping lift the cells off the plates. Can you check whether the floating cells are alive by trypan staining, it may be that you just need to let them sit down for a while.
Are you using the same amount of DNA and L2K for the control? What size plate/well are you transfecting?
Most transfections work best when the cells are about 50-70% confluent.
Have you tried other transfection reagents such as Fugene or PEI, which are much less toxic and work better (in my hands at least). You may also want to look at electroporation.
bob1 on Sat Nov 17 03:54:08 2012 said:
Your plasmid isn't the problem in terms of size, though the bigger it is, the harder it is to transfect; 8Kbp is not all that big. L2K is quite toxic by itself so it may be helping lift the cells off the plates. Can you check whether the floating cells are alive by trypan staining, it may be that you just need to let them sit down for a while.
Are you using the same amount of DNA and L2K for the control? What size plate/well are you transfecting?
Most transfections work best when the cells are about 50-70% confluent.
Have you tried other transfection reagents such as Fugene or PEI, which are much less toxic and work better (in my hands at least). You may also want to look at electroporation.
Hi bob1,
So I did a 24 plate with varying ratios of DNA:L2K and found that a 1:2 ratio works best for C3-GFP (about 80% efficiency), however, when it comes to my plasmid the efficiency about 5 percent
. As for the cell death, that really isn't an issue anymore using this ration. I also tried using PolyJet and that was actually worse in terms of efficency. So I guess now I need to make sure my DNA is actually real via a double digestion. Going to do a digestion today and make sure I have the right fragments. Otherwise I think electroporation will be the only way to go 
...THANKS!
Unfortunately different plasmids have different transfection efficiencies, I think this is mostly based on size, but it also seems to be dependent on the plasmid sequence to some extent and perhaps the promoters driving the gene of interest. Basically this means that testing a transfection with GFP will work only for the GFP plasmid, not your plasmid of interest.
If the digest comes out OK, it would probably be a good idea to also sequence the insert to make sure it is in frame and correct sequence.