So basically, I wanted to do a pilot expression using BL21(DE3) and my genes. The cultures(3 of them having different genes) were transformed and grown overnight in LB amp(50mg/ml). Next morning during my subculture step in fresh 10ml LB, I added double the amount of amp in one of my tubes by mistake.
First question:
1) Will my cells grow slower in that tube than the rest?
2) Does the extra amp affect protein expression, i.e. with loss of plasmid?
3) Can I compare the data from the eventual expression data(on a gel) I get from that tube with the rest?
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DeeDee
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Posted 22 April 2010 - 03:19 AM
Is yours a high copy number plasmid?? If so it should generate enough resistance to 50-100ug/ml amp concentrations.If the copy number is low,not all cells will cope with the increased amp dose so obviously the cell density will suffer and the overall extent of expression will be low.
Btw...i'm assuming you meant 50ug/ml instead of 50mg/ml(?)
Btw...i'm assuming you meant 50ug/ml instead of 50mg/ml(?)
Edited by DeeDee, 22 April 2010 - 03:21 AM.
