Hi everyone,
I need to expression of GFP with pET28 plasmid in E. coli BL21(DE3) plysE. Transformed cells have kanamycin and chloramphenicol resistance. Normally, only transformed cells should be present in the medium with these two antibiotics. However, I saw less fluorescence than expected under UV light on the petri plates (LB medium) prepared with IPTG and antibiotics (both antibiotics final concentration of 50 μg/ml). I then used the antibiotics separately for competent E. coli Mach1 cells without any plasmids in it. The kanamycin I used increasingly did not work and the competent Mach1 cells grew up. I prepared fresh kanamycin stock solution (50 mg/ml) and repeated with competent Mach1. They grew up again. I purchased the new kanamycin and repeated the experiment. They grew up again.
Finally, with the final concentration of 50 μg/ml, I used old and new kanamycin separately and inoculated C41, Mach1 and BL21(DE3)plysE (pet28-GFP transformed in) strains into solid and liquid media.
I attach some photos with last experiment:
A1: old kanamycin
A2: new kanamycin
M: Mach1, C: C41, P: BL21(DE3)plysE (pet28-GFP transformed in)
What could be the solution for this?
Thanks in advance!
Hi
It seems like your competent cells are contaminated since non-transformed competent cells (without plasmid) had colonies.
how old are your competent cells? Maybe you can prepare the new one and try the transformation step again. I usually use this protocol to prepare competent cell stock
http://2010.igem.org/wiki/images/b/bc/IGEM_2010_protocol.pdf
And have you checked the colony after transformation with Taq PCR, digestion check, or sequencing to confirm the GFP presence?
Thank you for the answer Nathania,
Transformed cells have remained at -80 degrees for over a year and I think they are still stable. When I use them for different experiments, I don't have any problems with the transformation process. The purpose of using competent cells was to use a non-plasmid strain. My problem is that strains without plasmids grow in a medium with kanamycin.
Since GFP has fluorescence under UV light, I didn't need control.
Dear M. Kajan,
The incubation time was 15-16 hours. is it too long ?
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