Bacteria growing on plate but not liquid media
Posted 09 July 2010 - 07:02 AM
I was having troubles earlier with ligation and transformation but now that I seem to have gotten that under control another issue has sprang up. The bacteria grow fine on an ampicillin plate (I ran a negative control of untransformed bacteria to unsure the amp was working correctly, I also ran a positive control to make sure the bacteria were alive and then of course I grew some of the transformant on an amp plate - the marker being ampicillin resistance). Transformants grew on the amp plate so I then proceeded to pick a colony and put it in 20ml of LB with 30micrograms/ml (600 mg). Nothing at all grew in the LB. I'm going to check to make sure I didn't botch the amp concentration somehow, but I really doubt I did. Any ideas guys? I assume the bacteria did transform since they lived on amp plates that should have otherwise killed them, and I'm relatively positive I managed to isolate a colony but maybe not. I don't feel like wasting another overnight to find out that my colony selection wasn't the issue so I'd like to trouble shoot a bit first.
Posted 09 July 2010 - 07:19 AM
Posted 09 July 2010 - 07:51 AM
Posted 09 July 2010 - 12:44 PM
Posted 09 July 2010 - 06:57 PM
Alternative, what kind of plasmid are you growing? Single copy plasmids and plasmids that express toxic protein do grow slowly. Some strains of E coli also grow slowly.
And sometimes there can be a problem with the shaking incubator. Ie the incubator is not holding the temperature at 37C
Posted 12 July 2010 - 05:58 AM
The strain of E. Coli is XL1 Blue so it should grow at a normal rate, it was grown over night and there were colonies by the next day (about 18 hrs).
Honestly, I'm not sure what single copy plasmids are (I'm assuming you mean from one colony and there's only one plasmid) but I believe it is.
Every time I've checked the incubator it's at 37C.
Posted 12 July 2010 - 08:27 PM
in multicopy plasmid, there maybe be 300-400 copies of the plasmids in each cell.
more than 2wk... yup it is probably the plates. Ampicilin does get hydrolysed.
Posted 13 July 2010 - 07:42 AM
Thanks for all the help
Posted 15 July 2010 - 08:12 PM