I am trying to transfect a plasmid in HEK293T and nHDF cell lines by using Neon electroporation system and have following 2 questions:
1) After successful transfection, what is the expected time required for the transcription of DNA into RNA (100bp) from U6 promoter in both above mentioned cell lines?
2) After successful transfection, what is the expected time required for the expression of a protein from DNA (1400 amino acids) via Chicken-beta-actin promoter in both above mentioned cell lines?
Overall, how much time it needs for the plasmid to reach nucleus and start transcription followed by translation using above mentioned promoters and respective downstream sequence of interest? Thank you.
If you are using non-viral plasmid (transient) 24 h time is enough for both, in case of viral method, you should wait for 48 h as virus need to be integrated and stabilized for proper expression.
Thank you Kumar. 24 hour is much longer for non-viral plasmid. Do you have idea about minimum time required for that? Actually my experiment is temporal-based so II need this kind of information.
You Can try to follow after 6 h for HEK293, but you need to do a time course to know exact optimal time according to your requirement. Other cell line I have never used, HEK293 is very robust . I am wondering you do you use electroporation as normal lipofection gives more than 90% transfection in HEK293. I am using XFect polymer from Clontech to transfect HEK, use 1:1 ration DNA:Xfect polymer, get more than 90% transfection with 10kB plasmid
Thanks Kumar! I am using lipofactamine2000 for HEK293T. I hope it can work well.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA