I am trying to stably transfect B cell line using Neon device, but during optimization of my experiments I noticed that most cells die after 72 hours (although I got about 60% positive cells with pEGFP-C1 plasmid within 1st 24 hours). Furthermore the remaining alive cells seems they are not growing any more. Does any one has experience with Neon (esp. stable cell line generation). In our Institute we have Amaxa device also, so do you people think I should shift to Amaxa or keep trying with neon? Thanks
I've never tried Neon, but I've had pretty good success in electroporating B-cell lymphoma cell lines with Amaxa. If it is a cell line I haven't tried before I generally try programs G-031, O-016, and X-001 with kit V to see which works best. You will need to balance between transfection rate and cell death when deciding on the best program. However, your chances of getting a stable cell line using electroporation without using a selectable marker are poor.
Actually this is another obstacle which you mentioned Michael about selection marker. My plasmid is retro-viral full length infectious clone, it is about 12 kb and doesn't have selection marker. I am working on inserting neo resistance gene; but so far I have no luck. This clone is very difficult to handle in ligation/cloning experiments as it is very prone to deletion/recombination in e coli, as it has 2 full length LTRs. We tried many different competent cells, but still need a lot of work to manipulate.
So far I am trying to co transfect my plasmid with other plasmid containing selection marker at ratio of 5:1 and try clonal selection hoping to find clone co expressing my plasmid of interest. I am not sure this is going to succeed, but no other choice until I could clone resistant marker in my plasmid.
have you experience with this type of co-transfection experiments for stable cell line generation?
Mohamed, the co-transfection should work, although the efficiency is lower. Try to increase the amount of plasmid, this should help.
I have a very good experience with Amaxa, you should try. For B lymphocytes you can use programs U-015 or Z-001. Recently I published a paper using different in house buffers for nucleofection. Maybe you could use one of them to see how it goes. http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0060298#pone-0060298-g008
Regarding the cloning protocol, try to incubate the tranformation and mini preps at 30 degrees, it helps with the deletion/recombination problem.
Leonardo, Thanks very much for providing me with your paper, probably I am going to try that if I went to Amaxa.
Regarding cloning protocol, For how long do you recommend incubation at 30 for transformation miniprep? at 37 I incubate for 1 hour.
In the transformation protocol, increse the incubation to 1:30h (after the heat shock) and incubate the plates overnight....if the colonies are small, incubate more time.
The mini prep inoculum can be grown overnight or more.
- Badges
- Science method