User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/21

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06/21/2013

Antibiotic (Hygro) Colony Selection

Mammalian vector V0200 (BD002 Backbone) has selection markers for Ampicillin (bacterial cell selection)and Hygromycin (mammalian cell selection) antibiotics.


Transgenic Cell Maintenance

Cell Type Genotype Medium Supplements
(required)
Maintenance
antibiotic(s)
U2OS (adherent) Flp-in T-REx, hygro-transfected McCoy's 5A 10% FBS, 1 % pen/strep 15 ug/mL blasticidin, 60 μg/mL hygromycin
U2OS (adherent) Flp-in T-REx, hygro-transfected McCoy's 5A 10% FBS, 1 % pen/strep 15 ug/mL blasticidin, 200 μg/mL hygromycin


  • Treat U2OS transfected cells with 200µg/mL of media for 5 days.
  • Cells were happy with new media for the first 2 days and only two small colonies remained on the edge of the plate after 6 days.
  • After 6 days growth, cells were again treated with Hygromycin-free media (U2OS media(p/s)).
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