Inoue help - again...

Michael J. Morales mmorales at acpub.duke.edu
Wed Feb 4 11:56:22 EST 1998


Robot-Boy wrote:
> 
> sorry to post again...had forgotten something else...
> 
> just some quick help. I'm going to try making competent cells the
> inoue
> way, and only had the recipe for making the cells, but not for the
> actual
> transformation. Do I just do the transformation the "regular" way ?
> ie.thaw on ice, add DNA, wait 20-30 minutes, heatshock, chill, add SOC
> and grow at 37 deg for 60 minutes, plate on selective media.
> 
I have found that you can do almost anything you want.  For routine 
subcloning (>10^8 colonies/ug), I add some of my ligation mix
(PEG is no problem) to 50 ul cells in a 1.6 ml microfuge tube.
20 minutes on ice, 30 sec @ 42 degrees, 2 min on ice, then add
450 ul SOC & let recover at 37 degree for 30 minutes.  This is
with DH5 alpha.

Hope this helps

Mike Morales



> I also wanted to ask what to use to bring the pH of the transformation
> buffer up to 6.7. I tried KOH and that didn't go well at all...I got
> some
> sort of precipitate (I guess some Manganese salt, since it has a
> purplish
> color). Should I use diluted KOH as opposed to 1 N KOH ?
> 
> thanks in advance,
> 
> Ed
>



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