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

From OpenWetWare

< User:Behzad Damadzadeh | Notebook | PcTF Subcloning in E-coli | 2013 | 06(Difference between revisions)
Jump to: navigation, search
(06/21/2013)
Current revision (18:35, 23 July 2013) (view source)
(Antibiotic (Hygro) Colony Selection)
 
Line 28: Line 28:
*Treat U2OS transfected cells with 200µg/mL of media for 5 days.
*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.
*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.
+
*After 6 days growth, cells were again treated with Hygromycin-free media (U2OS media(p/s)).

Current revision

PcTF Subcloning in E. coli Main project page
Previous entry      Next entry

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)).
Personal tools