One step transformation
"Mayer, Jorge ", 3637
J.MAYER at CGNET.COM
Fri Mar 3 19:23:06 EST 1995
In article <3itgh6$g3h at server.st.usm.edu>, Glen Shearer
<gshearer at whale.st.usm.edu> writes:
>Anyone tried the Chung et. al, PNAS. 86:2172-2175 (1989) one step
>transformation & storage of E. coli? As I recall, the authors use
>a solution of PEG + MgCl (called TSS) to resuspend log phase cells
>and get a reported 10^8 transformants per ug.
>I've tried this method with several strains and get at best 10^5
>transformants/ug with various pUC vectors.
>I'm curious if anyone has had this work in the 10^8 per ug range and
>if so, what is the secret technique.
>[gshearer at whale.st.usm.edu]
>I used modified methods of this and usually could get 10^8 per ug.
>I think the method was reported 1992 by Nishimura et al.
>If you can not find the protocol send me e-mail. I have a copy in my lab.
>One thing I want to mention is if you want to get more than 10^7 per ug DNA
>should use DNA less than 100 pg. If DNA is to much there will be a
>decrease of transformation because of saturation effect.
We have been using a method published in NAR 22, 2857-2858 (1994) with
great success (2x10^9cfu/ug). It's simplicity is incredibly attractive; in
principle it's just harvesting the cells at OD 0.9 instead of early-log.
Jorge Mayer <j.mayer at cgnet.com>
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