Competent cells- just won’t work!

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    • #13672
      weenold
      Participant

      I have made both electrocompetent and chemically competent cells of several E. coli strains in order to transform them with a plasmid. One strain worked when I made electrocompetent cells, but for the other strains I am getting no colonies at all (2 pathogenic strains and one lab strain). I have made a new plasmid prep, cleaned it up, used pre-warmed SOC, but I am still getting nothing. I have used a control of pUC19, known to be clean, and didn’t get any colonies either. I have also checked the antibiotic concentration. Any ideas what could be the problem? I have made competent cells in the past, which have worked, although they’ve never been super-competent. I’m getting a bit fed up so any comments would be appreciated!

    • #100930
      JackBean
      Participant

      does your transformation work? Are you able to get colonies after transformation of any (old) cells?

    • #100932
      weenold
      Participant

      Well the electroporation worked for one strain. As for the chemical transformation, I’m not sure, since I have recently had to move from a water bath to a heating block, perhaps I am not achieving the correct temperature.

    • #100936
      JackBean
      Participant

      yeah, I see…
      well, then probably look to all your chemicals you’re using, whether they are OK. Are the cells able to grow on agar without antibiotics?

    • #100970
      weenold
      Participant

      Yeah, the cells grow fine when plated directly onto agar (before transformation). Unless I am killing them in my electroporation/chemical transformation. Maybe my technique of making competent cells is flawed. I try to keep them cold all the time, but perhaps I am too rough with their resuspension? I thought I’d achieve at least a few colonies though!

    • #100975
      JackBean
      Participant

      yeah, that could be it. Are you resuspending by pippeting?

      Are the cells viable before transformation but after being-made-competent?

    • #100986
      canalon
      Participant

      You say that you are using some natural isolates there are a few other things that can prevent transformation on those in spite of perfect reagents:
      – restriction/methylation activity. your new DNA might not be recognized by the methylation/restriction system of your recipient strain and is quickly degraded.
      – presence of incompatible plasmids in your cell that will prevent the replication of the new one
      – expression of capsule or similar membrane alterations that will make your bacteria much less likely to take up DNA

    • #101010
      weenold
      Participant

      Yes I am resuspending by pipetting.

      Yeah you’re right, these isolates aren’t as amenable to being made competent, however, I’ve managed it in the past. I just checked the pH of the milli-Q water I was using and it’s apparently pH 4! Could this be the problem?

    • #101012
      canalon
      Participant

      MilliQ water is basically pure H2O, and so has no buffering ability, and the dissolution of C02 in water will acidify it. To increase pH just autoclave it, all dissolved gases will go, but that won’t last long as the atmospheric CO2 will dissolve back.

      So in short your ultrapure water will always be acidic, and that is always the case, so it is probably not the problem here. Since one of your starins did work, it seems that the method is fine. I would suspect your cells. HAve you tried another method (Rubidium chloride is supposed to make chemically competent cells almost as good as electrocompetent one, and with much less work)

    • #101121
      weenold
      Participant

      Hmm, yeah apparently you can’t read the pH of ultrapure water with a pH meter and the fact it’s 18 ohm should be sufficient proof that the pH is 7? At least when it comes out of the machine.

      I’ve tried rubidium chloride and calcium chloride both unsuccessfully. ARGH!

    • #101134
      canalon
      Participant

      Have you checked if there are already plasmids in your strains. Maybe you have a compatibility problem?

    • #101257
      weenold
      Participant

      As far as I am aware they don’t contain any incompatible plasmids.
      I wonder if it is because I tried to scale down the competent cell protocol in order to not have excessive numbers of cells at the end (i.e. 40 tubes when I only need to succesfully transform them once). Could it mean they are more likely to rise about 4oC during the procedure?

      Also, which is more important – gentle resuspension or keeping cold? I find gentle resuspension difficult at times because it can take so long. In the protocol it recommends "gently swirling" the tubes, but I’ve tried this and I’d be there all day if I took this approach. Any tips on "gentle resuspension", is using a pastette a better idea than a normal pipette tip? I can only assume I am doing something seriously wrong at some point.

    • #101263
      canalon
      Participant

      Don’t know about the scaling down, never tried it. But considering the cost of reagents vs the time taken by repeated failure, maybe worth trying preparing 40 tubes… or even 20.

      However I am surprised that you have problem resuspending the cells. My experience is that when washed in a lot of water (and I do not know why) it is quite hard to get a pellet that is solid. they tend to resuspend a bit too quickly.

      A pasteur pipette will be a bit gentler (larger opening), or flame it to form a glass sphere at the end and gently use that to mix your suspension.

    • #101407
      kimsmarkin
      Participant

      Electroporation has worked for the same stem. Regarding the chemical transformation, I am not sure when I recently had to move from one water bath to a heating block, maybe I did not have the right temperature.

    • #102766
      pavanibeesetty
      Participant

      Hi,
      I have been using Zymokit to make competent cells and I have the same problem as u mentioned. I have been trying to make competent cells of a uropathogenic strain of E.coli and the cells I get are not that competent. But i was able to successfully transform them with pUC19 but am not able to transform with my Kan R plasmid. The cells do not have any other plasmid. I think maintaining 4C is very imp during making this competent cells as similar trial which i performed before didnot work (which i think because at some point the temperature of the competent cells is increased to above 4C while prep). But the second time where i took great care of temp I was able to transform atleast my positve control (pUC19).
      And may be mixing the cells gently might also have some effect on the cells.
      One more suggestion given to me by the tech support guy of Zymocompany is to grow the cells in Zymobroth with a very low initial OD. (I donno if any one is using this kit. But i just wanna help the people who has faced my situation.

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