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E.coli culture

  1. Apr 16, 2004 #1

    Monique

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    Ian? What happens if you leave E. coli's out to grow on a AMP/LB plate overweekend? :frown: I left my plate in the stove and they are REALLY important clones and I don't have the key to the lab!! :eek: They'll live right?
     
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  3. Apr 16, 2004 #2

    iansmith

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    It should live only if your plates were inverted and your incubation does not go beyond 96 hours. If your plates were not inverted, the plates migth dry out and it migth kill your bacteria. Be sure you are in early morning and restreak your colonies on fresh LB. Do you have frozen permanent or any sort of backup (e.g. plate in the fridge)?
     
  4. Apr 16, 2004 #3

    Monique

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    96 hours huh? *relief* this wás actually my backup, I plated 13 different most promising colonies on this one plate so that I could plate them out once the result of the colony pcr on 66 colonies came back.. out of the 66, three finally came out with the right thing.. which were on that plate :rolleyes: (and yes, I did a blue/white screening) it's a really difficult construct so I wouldn't want to do the whole thing from start again :smile:

    What are exactly those satellite colonies that come up if you grow them too long? I know the amp gets broken down by the resistant colony, but do those satellite colonies just come dropping out of the air?
     
  5. Apr 16, 2004 #4

    iansmith

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    Amp inhibits the growth of bacteria but does not kill them. By the time amp is broken down some bacteria have survive the starvation and start growing. This is why satellite colonies are smaller. The satellite colonies will only appear during your first round of selection. Not satellite is present after you select and streak for isolation your desired clones.
     
    Last edited: Apr 16, 2004
  6. Apr 16, 2004 #5

    Monique

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    oh really, I didn't know that..
    thanks Ian :biggrin: :smile:
     
  7. Apr 16, 2004 #6

    Moonbear

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    Monique, have you considered calling somebody who has the lab key to let you in, or, alternatively, who can take your plates out for you if they are in the lab this weekend? On most campuses, you can call security to let you in if you usually are supposed to have access and have misplaced your key (or locked it in inside the lab, as I've done to myself several times). Just calling the lab and leaving a message usually does the trick...I've never been in a lab where SOMEBODY wasn't working over the weekend ;-) I know that doesn't directly answer your question, but it may solve the problem nonetheless. Or email your PI. Everyone has done something "forgetful" in the lab at some time in their life, and this is a pretty minor one, so I'm sure someone will take mercy on you and let you in to retrieve your plate. Good luck with those colonies.
     
  8. Apr 17, 2004 #7
    What are you guys talking about ?
     
  9. Apr 17, 2004 #8
    I think what's going on is that Monique left some cultured E.coli (a bacterial) in a stove, and can't access them for the weekend, and was wondering if they would survive. Apparently they will as long as the plate is inverted, so the colony on the plate stays moist.

    AMP here stands for ampicillin (an anti-biotic) and not adeno-mono-phosphate (a metabolite). LB is an agar plate, in which E.coli is grown on.Something like that anyway :p

    Anyway, a question on my part - Can AMP kill bacteria or will it only stop growth? Or is it only able to kill cells that are actively dividing?
     
  10. Apr 17, 2004 #9

    Monique

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    :smile: I called but there's noone there :rolleyes:
    I could go there, but that'd cost me 3 hours total traveling time.. and I'd have to make a fool of myself searching through the whole academic hospital for a guard with a key.. just for a silly plate :redface:

    As Ian said, I'll restreak them on monday :)
     
  11. Apr 17, 2004 #10

    Monique

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    Good question, what is the molecular basis on which ampiciline works? From Ian's statement I understand it is biostatic and not biocidal.
     
  12. Apr 17, 2004 #11

    iansmith

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    The beta-lactam group of antibiotics is only bactericidal on growing cells. These antibitotics inhibits the formation of peptidoglycan cell wall. Once the cell wall is destroy the bacteria lose their shape and cannot regulated the desired osmotic pressure. The cell lyses due to acumulation of water in their cytoplasm. In vitro, you can stop the lysis of gram positive by using special media and you end up with bacteria lacking a cell wall and the bacteria are all round. Cells that are not actively growing will not be killed.
     
  13. Apr 17, 2004 #12
    Monique, what are you doing with the E.coli culture? Are you growing some that are resitant to amp?
     
  14. Apr 17, 2004 #13

    Monique

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    No, it is a non-pathogenic strain of E. coli which normally is not able to grow in the presence of ampiciline. What I made is a certain contruct in a viral vector, a piece of dna, which the bacteria have taken up. In this contruct there is a piece of DNA that contains the restistance gene against amp. So only the bacteria with the construct will be able to grow. I can later isolate this vector with the insert, a plasmid, from these bacteria.

    So the construct that is made can either contain a gene or portions of DNA that regulate genes. The bacteria just serve to amplify it, so that it can be isolated and put back into another celltype to evaluate its effect.
     
  15. Apr 17, 2004 #14

    iansmith

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    In other words you are doing cloning with probably DH5 alpha with a pUC derivative :wink:
     
  16. Apr 18, 2004 #15

    Monique

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    Close, cloning with DH5 alpha and pGEM(-T) (among others: pGL3, pcDNA3.1) :)
     
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