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CAS:31282-04-9
Appearance:Off-white to yellow solid
Storage:Powder : 2-8℃, 2 years; In solvent(mother liquid): -20℃, 1 month; -80℃, 6 months
Purity:≥90%
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Examples of using this product（for reference only）
In Vitro:
Cell (HEK293T cells; 100-400 mg/mL Hygromycin B):
In brief, the plasmid DNA was isolated from the TOP10 F' Escherichia coli containing pcDNA3.1 /Hygro (+)-ASPH using plasmid miniprep kit according to the manufacturer's protocol. The plasmid was then linearized using FspI digestion. HEK293T cells (2×105 cells/well) were seeded in 6-well cell culture plates to give optimal confluency (70%) at the time of transfection. Transfection was carried out using 4 μg of plasmid DNA and 8 μL of transfection reagent. After three days, the medium was replaced with a fresh medium containing 100 mg/mL hygromycin B (Solarbio Science & Technology, China). The plate was fed with fresh selective medium every 4 days and was screened for resistant cells remained in different parts of the plate. The detected resistant cells were monitored for 4 weeks to spot separated subclones with the ASPH expression potential. Five selected subclones were carefully scraped off the plate using sterile scrapers and transferred to separate flasks. After 2 days, the cells were trypsinized and redistributed in the flask. The mRNA expression level of each flask was evaluated as described below (subsection qRT-PCR) to select the subclones with the highest ASPH expression. Then, the concentration of hygromycin B was gradually increased in the selected flasks to reach 400 mg/mL after 4 weeks of culture.
Reference：
Bakhtiari H, Palizban AA, Khanahmad H, Mofid MR. An innovative cell selection approach in developing human cells overexpressing aspartyl/asparaginyl β-hydroxylase. Res Pharm Sci. 2020 Jul 3;15(3):291-299.doi: 10.4103/1735-5362.288436.
