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Thread: Making cells supercompetent in the lab

  1. #1 Making cells supercompetent in the lab 
    Forum Freshman
    Join Date
    Mar 2010
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    Currently I am trying, and largely failing, to make XL1-Blue MRF' Kan cells supercompetent - in the region of 10^9 transformants/ug DNA when tested with pUC19. I have achieved 7x10^8 transformants/ug DNA, but when testing multiple aliquots discovered that the efficiency ranged from 10^5-10^8 from one batch of cells, which is not much use to me as I obviously need to know that the efficiency of my entire set of aliquots is as similar from tube to tube as possible.

    I am following this procedure exactly as it is written here

    Sol. 1 - 600ul 1M MgCl2, 3ml 1M CaCl2, 12ml 50mM MES made up to 60ml with dH2O
    Sol. 2 - 120ul 1M MgCl2, 600ul 1M CaCl2, 2.4ml 50mM MES, 3.6ml 50% glycerol made up to 12ml with dH2O

    1. Set up o/n culture of XL1-Blue MRF' Kan from single colony streaked from glycerol stock, grow at 37C, 250rpm
    2. Subculture 2.5ml o/n into 100ml LB broth supplemented with 1.5ml 1M MgCl2 and grow to OD600 of 0.5 at 37C, 250rpm
    3. Rack sufficient microfuge tubes and place at -80C for aliquoting cells
    4. Harvest cells at 4C for 10 minutes at 2000rpm
    5. Remove supernatant and resuspend cells in 60ml ice cold Sol. 1. by swirling gently on ice. Incubate on ice for 20min.
    6. Harvest cells at 4C for 5 minutes at 2000rpm
    7. Remove supernatant and resupend cells in 12ml ice cold Sol.2 by swirling gently on ice.
    8. Aliquot cells in pre-chilled microfuge tubes on ice, immediately snap-freeze in liquid nitrogen and store at -80C

    Can anyone see an area of this procedure which could be improved/altered in any way to improve my cell efficiency? I'm just looking for a bit of external input now as I have done this so many times that I think I must be missing something and overlooking a potential problem.

    The variables that I have changed so far are to closely monitor the OD600 instead of determining it 'by eye', and ensuring I am using an OD600 of 0.5 exactly, and I have also tried using colonies from a freshly streaked plate as opposed to one which has been lingering in the fridge for a week or two. I have also ensured all my glassware is clean by autoclaving dH2O in it, rinsing it out, and adding media prepared in equally clean glassware prior to use.

    I would, if I really had to, be happy with using 10^8 transformants/ug DNA cells as long as I could be sure that all my aliquots were this competent, however I really am looking for 10^9 in an ideal situation.

    I'd appreciate any input in this, however small an alteration might seem - I need these competent cells for bacterial 2 hybrid system and have to propagate my plasmids for that in these cells as well (they're toxic to other strains) so I can't go anywhere with the procedure until I get the cells working. I could buy them in, but spending so much on cells when the cash is not exactly readily available seems unecessary when I believe it is possible to do it myself...


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