I did a ligation and transformed into dh5a cells and grew at 37C. I checked the plates 14 hours into incubation, there were either no colonies grown at that time or i missed them. Anyways i left them in there because i forgot about them and remembered they were in there and went to dispose of them about 36 hours into incubation and there were some huge colonies on the plates (about 5-10 a piece). Some coloneis had the little colonies around them, im assuming were satellite colonies, and some didnt. My question is, are the colonies still good for being incubated that long. I will test to seee if they contain the appropriate plasmid later but i want to know if something bad happens to them for being incubated for so long, the ligation probably worked so i hope they are ok, at least i think it worked. I did have 50ug/ml carbenicillin on the plates and they were stored in 4C for about 10 days before use. thanks for you help.
-shimshady-
shimshady on Mar 22 2009, 05:35 PM said:
I did a ligation and transformed into dh5a cells and grew at 37C. I checked the plates 14 hours into incubation, there were either no colonies grown at that time or i missed them. Anyways i left them in there because i forgot about them and remembered they were in there and went to dispose of them about 36 hours into incubation and there were some huge colonies on the plates (about 5-10 a piece). Some coloneis had the little colonies around them, im assuming were satellite colonies, and some didnt. My question is, are the colonies still good for being incubated that long. I will test to seee if they contain the appropriate plasmid later but i want to know if something bad happens to them for being incubated for so long, the ligation probably worked so i hope they are ok, at least i think it worked. I did have 50ug/ml carbenicillin on the plates and they were stored in 4C for about 10 days before use. thanks for you help.
I suspect that the colonies could be something else could be contamination. You say that you cant see any colonies after 14 hrs but did after 36 hours. Do a quick colony PCR to check for the plasmid. But really its alot of trouble. Just plate a new one.
-MaggieRoara-
shimshady on Mar 23 2009, 12:35 PM said:
I did a ligation and transformed into dh5a cells and grew at 37C. I checked the plates 14 hours into incubation, there were either no colonies grown at that time or i missed them. Anyways i left them in there because i forgot about them and remembered they were in there and went to dispose of them about 36 hours into incubation and there were some huge colonies on the plates (about 5-10 a piece). Some coloneis had the little colonies around them, im assuming were satellite colonies, and some didnt. My question is, are the colonies still good for being incubated that long. I will test to seee if they contain the appropriate plasmid later but i want to know if something bad happens to them for being incubated for so long, the ligation probably worked so i hope they are ok, at least i think it worked. I did have 50ug/ml carbenicillin on the plates and they were stored in 4C for about 10 days before use. thanks for you help.
The large colonies should be OK, but the small satellites aren't worth worrying about.
-swanny-
