Hi everyone!
We are trying to express and purify the M-MLV reverse transcriptase construct from the Reclone Latin America Starter Kits in E. coli BL21, but we are struggling to see clear induction bands on SDS-PAGE.
We tried two different induction conditions so far:
- First attempt (left gel): Overnight at 30°C (since our incubator cannot cool down to 18°C).
- Second attempt (right gel): Room temperature (20 - 23°C) in an open shaker overnight, testing different OD_600 points (0.3, 0.6, 1).
As you can see in the attached image, while other enzymes like Bst and Phusion show clear, distinct overexpression bands, the M-MLV non-induced (nd) and induced (ind) samples look almost identical, with no strong bands indicating robust protein production.
Has anyone in the community faced similar issues expressing this specific M-MLV? Are there any specific optimization tips regarding induction temperature, media, or induction times that could help improve our results?
Any advice would be greatly appreciated.
Thank you!
Marie.