HI everyone,
We've recently starting doing S2 cell work in our lab again and we have been having some issues getting transient transfections to work.
We are working with standard S2 cells (not R+). We have made all fresh media and have ordered a new transfection kit (one that we have used in the past). We also are transfecting in parallel a published construct from our lab we know that has worked in the past as our "positive control". I have grown the control vector up from a -80 E. coli glycerol stock to have freshest DNA available. I also checked our induction reagent (CuSO4), and that is still blue and good to go. All plasmids were isolated using a midi-prep kit (2 year old Qiagen)
I have been reading online about the typical troubleshooting one can do for S2 cells and the only thing that could be an issue is the DNA concentration we got from the midi prep kit . We only retrieved ~100 ng/ul of DNA in a volume of 350 ul. Could the DNA be too dilute or impure? The 280/260 ratio was just above 1.8 for each sample when we nano dropped the plasmids.
Are there any other atypical problems I should be considering?
Thanks for your feedback!
I woulda agree that low-yield DNA preps may not work well in transfections. If the DNA yield is 50% of normal it should still work, but if its only 10% I would repeat the prep.
- Badges
- Science method