Nov 03, 2025
Blunt-End Ligation of Synthetic Lethal 7 Gene into pMAL-c2x Vector and Its Protein Expression in E. coli
- Taohsueh Liao1
- 1Tmkang University Life Science, No.151, Yingzhuan Rd., Tamsui Dist., New Taipei City 251301, Taiwan
- Taohsueh Liao
Protocol Citation: Taohsueh Liao 2025. Blunt-End Ligation of Synthetic Lethal 7 Gene into pMAL-c2x Vector and Its Protein Expression in E. coli. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6zeyrgqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 02, 2025
Last Modified: November 03, 2025
Protocol Integer ID: 231348
Keywords: end ligation of synthetic lethal, gene into pmal, synthetic lethal, end ligation, protein expression, corresponding gene, gene, protein
Abstract
To express the Histone synthetic lethal 7 protein, the corresponding gene was cloned into the pMAL-c2x vector using blunt-end ligation and transformed into competent E. coli BL21(DE3) cells.
Image Attribution
Figure 1: Protein expression was confirmed through cell disruption and SDS-PAGE analysis. The second band from the top corresponds to the target gene. 95kDa.
Troubleshooting
Method
Initially, 5 mL of LB medium was inoculated and cultured for 22 hours at 37°C, reaching an OD600 of 2.41.
Subsequently, 4.3 mL of the cultured broth was mixed with 95.7 mL of LB medium supplemented with sorbitol and ampicillin (10 mg/mL), resulting in a final volume of 100 mL and an OD600 of 0.6.
Protein expression was induced by adding 0.8 mM IPTG, followed by incubation at 30°C for 22 hours.
After induction, 1 mL of bacterial culture was transferred to an Eppendorf tube and centrifuged at 5000 rpm for 7 minutes at 4°C using a refrigerated centrifuge.
Protein expression was confirmed by SDS-PAGE analysis (see Figure 1). Lactose was not used for induction due to insufficient protein yield observed in preliminary trials.
Figure 1: Protein expression was confirmed through cell disruption and SDS-PAGE analysis. The second band from the top corresponds to the target gene. 95kDa.