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Bacteria growing on plate but not liquid media


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#1 Shadowstep33

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Posted 09 July 2010 - 07:02 AM

Hey guys,

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.

-Gabe

#2 phage434

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Posted 09 July 2010 - 07:19 AM

Well, this is probably a typo, but you did say 600 mg in a 20 ml tube. That would be 300x - 1000x too much, if you really meant it.

#3 Shadowstep33

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Posted 09 July 2010 - 07:51 AM

oops yeah typo, i used 600 micro grams. 6 micro liters of a 1 ml vial that had 100mg of amp.

#4 molstudent

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Posted 09 July 2010 - 12:44 PM

i had this issue with many plasmids and haven't figured it out yet. all i was doing was plasmid amplification too. i would like to know the answer to this if anyone knows it..

#5 perneseblue

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Posted 09 July 2010 - 06:57 PM

how long did it take for the cells to appear on the Amp plate? Might these colonies be false positives? Amp does degrade, so a 2 wk old plate or a plate kept incubating for over a day or two, may not have sufficient Amp to give selection. Amp doesn't kill none dividing cells, so once the Amp concentration falls below a certain concentration, some Amp sensitive cell can start growing

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
May your PCR products be long, your protocols short and your boss on holiday

#6 Shadowstep33

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Posted 12 July 2010 - 05:58 AM

The Amp plates are probably more than two weeks old so I'm going to make some new plates just in case that is the issue. I doubt the colonies are false positives though because I ran non transformed cells on an amp plate from the same sleeve as a negative control and nothing grew on that plate.

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.

#7 perneseblue

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Posted 12 July 2010 - 08:27 PM

a single copy plasmid, is a plasmid that propagates as a single molecule per cell. Thus there is only one plasmid/BAC in each cell.

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.
May your PCR products be long, your protocols short and your boss on holiday

#8 Shadowstep33

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Posted 13 July 2010 - 07:42 AM

alright so I got it working. Honestly, I'm not sure what I was doing wrong. Maybe I was unlucky and picked a colony that was a false positive, someone in my lab also mentioned to me there's a phage that kills bacteria when you get a contamination of it. At any rate, it works now. I did make new amp plates though and the cells grew fine on those plates so I'm pretty positive that I have the right cells.

Thanks for all the help :)

-Gabe

#9 jangajarn

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Posted 15 July 2010 - 08:12 PM

but listen to pernesblue. Do away with ampicillin and use carbenicillin, the more stable variant of amp (same chemical structure and all)




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