meyerdj at phibred.com, Dr. David J. Meyer <meyerdj at phibred.com> wrote:
>Thanks are due Francis Ouellette for keeping this group running!
>Quick question: for how long at 4 degrees C are 5-FOA plates still
>counterselective for URA3?
>Also, how many generations of pregrowth under conditions nonselective
>for URA3 are required for a reasonable recovery of transformants if
>you are trying to shuffle out a CEN-based plasmid? And can I still use
>medium for the pregrowth which is selective for other markers (plasmid
>and chromosomal) as long as the medium contains uracil (say in the
>concentration found in SD)?
>Thanks in advance!
>David J. Meyer, Ph.D.
>Pioneer Hi-Bred International, Inc.
>7300 NW 62nd Ave.
>Johnston, IA 50131-1004
>Email: meyerdj at phibred.com
Here is a summary of replies, authors names deleted if they replied by
"We've used FOA plates that were a year or so old (kept at 4 degrees)
without any problem. So I think they have a very long shelf life. In
fact, we wrap our plates individually with paraflim when we store them
to keepthem moist longer--they seem to dry out before they lose in any
Pregrowing on other selective media works find--we replica plate
directly from SD-leu to FOA and it works fine. However, we do use
the pRS series of plasmids, which supposedly are not as mitotically
stable as other CEN vectors. I do prefer one round on YEPD. We
typically patch transformants onto a selective plate, replicate to
YEPD, then the next day replicate (or streak from the patches) onto
FOA. Works great."
"I have used FOA plates kept at 4oC for at least six months, without
any problems, to select against URA3 from a CEN plasmid.
I usually inoculate non-selective medium (in my case YPD) with the
minimum amount of cells and let it grow for at least 36hr (they are
almost saturated at that point). Then I plate 50 ul of a 1/1000
dilution on FOA plates (in 100mm diameter dishes). I haven't done any
quantification, but this gives me plenty of colonies. I think you can
use medium that is selective for the markers you want to keep for the
pregrowth, and probably in the FOA plates too (I haven't done it
myself, but it should work)."
Thanks to all who replied!
David J. Meyer, Ph.D.
Pioneer Hi-Bred International, Inc.
7300 NW 62nd Ave.
Johnston, IA 50131-1004
Email: meyerdj at phibred.com
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