Stored bacteria lost plasmids? - (Feb/06/2009 )
Quasimondo on Feb 10 2009, 02:04 AM said:
cellcounter on Feb 10 2009, 06:00 AM said:
You can also spot the plasmids on 3M paper, put it in a plastic sheet, and keep a notebook that way. Our neighbour lab supervisor has an album of such plasmids.
She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.
She has made multiple marked spots on each 3M sheet, and whenever someone requests plasmids, she just cuts a sqaure and sends it. She has been keeping it this way for over a decade now.
Yeah, so creative, plasmids can be easily stored at RT.
@KatieB: I have a reference for you. This sometimes happens in some labs and I also got this problem before, a little bit less serious, just the plasmid yield was very low. In this paper, they grew the cell in the media containing the excess concentration of ampicillin, since this would select the cells containing high number of plasmid copy. Hope this helps
.That is really interesting! thank you.
-KatieB-
Hi everyone, thank you for all your comments and help. I have managed to get everything working again, I made fresh ampicillin and all fresh media and still had afew problems with making fresh clones. so went back and got a fresh vector kit (my other one was quite new),. after doing this my cloning is working again and i've now brought up stocks from freezer of old plasmids which are all growing fine and working perfectly on realtime PCR 
Therefore, basically I must have had a multitude of problems with both media and vector kit. advice - never rely on expiry dates on kits!!!
Anyway, thanks for everything,
Katie
-KatieB-
KatieB on Feb 22 2009, 03:23 AM said:
Hi everyone, thank you for all your comments and help. I have managed to get everything working again, I made fresh ampicillin and all fresh media and still had afew problems with making fresh clones. so went back and got a fresh vector kit (my other one was quite new),. after doing this my cloning is working again and i've now brought up stocks from freezer of old plasmids which are all growing fine and working perfectly on realtime PCR Therefore, basically I must have had a multitude of problems with both media and vector kit. advice - never rely on expiry dates on kits!!!
Anyway, thanks for everything,
Katie
Therefore, basically I must have had a multitude of problems with both media and vector kit. advice - never rely on expiry dates on kits!!! Anyway, thanks for everything,
Katie
Congratulation, nice to hear that ^^. I also agree that "never rely on expiry dates on kits" because recently I found out one of my Kit was overdued for 6 months but thank God, it still worked well, lol
-Quasimondo-