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Whaaaaa my cells didn't grow:(

XiraXira Member Posts: 437
in Off-Topic Discussion
So I wake up at 5 frikkin AM so I can make some cells electro competant for a knockout and what do I find when I get to the lab at 6 !@##$!@ing am? Seems Salmonella Thypi dosen't like the pkd46 plasmid....Or mabey it wasn't selected for or mabey the plates weren't Amp+ or mabey it's got a restriction enzyme that's chewing it up or something. Argh. Annoying.

Comments

  • SerienSerien Member CommonPosts: 8,460


    Originally posted by Xira 
    Seems Salmonella Thypi dosen't like the pkd46 plasmid


    aw c'mon, we all know that

    lol..just kidding

    are you majoring in some sort of science?

    or are you in that field?

    i hope you are....instead of just making plasmaidoc +lierbadjvielad = hypoglocymeahsodflfghje!

    image

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    http://www.facebook.com/murtb

  • BrotherAlphaBrotherAlpha Member Posts: 7

    Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).

    Then again, you might just be unlucky.  Try again tomorrow!

  • DewfireDewfire Member Posts: 89


    Originally posted by BrotherAlpha

    Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
    Then again, you might just be unlucky.  Try again tomorrow!


    Yea thats what I always try too... image

    Dewfire Gnome Paladin EQ2
    Retired: AO, DAOC, EQ, SWG

  • BigPeelerBigPeeler Member Posts: 1,270


    Originally posted by BrotherAlpha

    Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
    Then again, you might just be unlucky.  Try again tomorrow!


    ... my thoughts exactly image

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  • XiraXira Member Posts: 437


    Originally posted by BrotherAlpha

    Transfecting plasmids can be both frustrating and time consuming, mainly because it doesn't always work, especially when you're inexperienced.  I assume that the plasmid you're trying to insert contains an AmpR gene for selection.  It might just be that your cell concentration at transfection was insufficient (remember that only a small proportion of cells are likely to be transfected, depending on the plasmid size and cell permeability).   Another thing worth trying is heat-shocking the cells, or performing some other permeabilisation step prior to transfection (sometimes works wonders).
    Then again, you might just be unlucky.  Try again tomorrow!


    I think it's dat last one. Or mabey it was grown at a plasmic-destroying temprature(temp sensitive replicon).

    I didn't do the transfection myself...Suffice to say that if _I_ had done it it would have worked:) Useing electroporation. The postdoc in charge of the lab said she'd take care of it and proceeded to make the transformants useing the "good enough(tm)" Ca-CL+ method....This produced transformants that grew on what (We think) was an Amp plate. Just that when put into an Amp+ culture to prepare them for further electroporation they all died. As for what's wrong...Eh...Well perhaps the plate wasn't really Amp, or perhaps the plasmid she was useing didn't actually have plasmid in it.

    After some investigation it SHOULD work(Nothing in Salmonella prohibits the Red-46 gene disruption protocol) and it's even been done successfully in other strains...As to what's wrong no clue.

  • MegaWolfMegaWolf Member Posts: 425

    Have you tried transconducting the amp plate? It may change the protolithocell number to 4.98 instead of 5.12. That should stablize the co-refronsire making the operation not only safer but more successful.

    image

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  • XiraXira Member Posts: 437
    *throws a sallabaloon at Megawolf*

  • BrotherAlphaBrotherAlpha Member Posts: 7
    Lol! Sounds like there's too many "if's" in that procedure.  If's aren't good in transfection ;)

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