I am trying to transfect lymphoblast cells and am achieving very low transfection efficiencies (~1%). I have used electroporation using the Lonza nucleofector kits V (program B-23/G-30) and kit T (G-16). I have also tried Lipofectamine 3000 with no success.
Any suggestions will be appreciated - thanks!
Hey Holly,
have you tried good old "pure" electroporation yet?
We achieve very high efficiency, however, a death rate of about 30-50 %, depending on the conditions with it.
Bests,
Christopher
Hello
Yes you can try with Electroporation or Calcium phosphate methods . The electroporation much better than lipofectamine but you need lots of cells (10 million per transfection!).
Good Luck
http://www.biomedcentral.com/1472-6750/10/9
http://www.ncbi.nlm.nih.gov/pubmed/15121168
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1473967/
Hi Holly.
I am currently trying the same. I am using the Nucleofector 4D (lonza) with the SF solution and program EW-113. when using Lonza control plasmid (pMAX) I normally get up to 50% GFP+ cells but when I use my plasmid (twice in size than pMAX) I dont get more than 1% of GFP+ cells. Have you got any success with this?
Hi Diego,
Our lab has recently received the 4D system to use as a demo. I am planning to try it out possibly next week so I can let you know how I get on.
I asked lonza for the programs I should use. They said I can check DS130, DT130, DS132 and DT132. I asked to a wormate from my previous lab and she recommended to use the EW113. In two experiments with the pMAX plasmid, EW113 gave the best results but yesterday I tried to use my plasmid of interest and I don't have more than 1% of GFP+ cells. What kind of plasmid are you trying to nucleofect?
I'm transfecting the pcDNA3 vector with a 15kbp insert. Maybe try changing the concentration of plasmid?
I have tried to transfect 200000 cells with 0.4 0.6 0.8 1 1.5 and 2 ug (Lonza recommends using from 0.4 to 0.8 ug) in the strips and nothing worked. I'm trying now to use an endotoxin-free maxiprep kit as the plasmid I used for the experiments wasn't endotoxin free.
We currently have EXACTLY the same problem with LT cells... No way to make this Amaxa 4D work except for their nice pGFP CMV promotor plasmid that is... USELESS!
I think this should undergo viral. They use their monopole to sell you something not working while pretending it is... The previous verison of their nucleofactor worked better...!
We cannot even obtain from them the electric field sequences they use...
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