Library plasmid rescue in two-hybrid sysytem

Christian Fritze cfritze at ix.netcom.com
Fri Aug 14 09:57:12 EST 1998


For those of you doing these rescues routinely, it may be worth tracking
down the references for the "double-zap" electroporation protocol (Brent
lab?):

Pick up a bit of yeast colony on a pipette tip
Mix gently with electrocompetent KC8
Put mixture into electroporation cuvette
Zap once, wait 30 secs, Zap again (different settings for the pulses)
Plate mixture on LB+Amp or M9 selective plates

I'm no longer a yeast worker, but perhaps this note will prompt others to
post their protocols for this. The most important parameter for success in
my hands was to be sure that the yeast plates weren't too old (< 1 week, and
always kept at room temp. or above). Good luck.

--
Christian Fritze, Ph.D.
Director of Technology Transfer
Berkeley Antibody Company (BAbCO)
www.babco.com
christian at babco.com
(510) 412-8930, -8940 fax
Frederik Boernke wrote in message
<35D461E4.345A at nospam.ipk-gatersleben.de>...
>-- gmclab221 wrote:
>>
>> Hello all. I have been trying to retrieve  positive prey plasmids from
>> yeast EGY48 after a two-hybrid library screen. I am attempting to
>> electroporate KC8 bacteria which are -trp deficient with a yeast DNA
>> mini-prep in order to rescue the plasmid but I cannot get any
>> transformants. I have checked my elctrocompetent cells with a control
>> plasmid and they are fine. I also am plating on less stringent LB/Amp+
>> plates initially instead of minimal media plates in order to improve my
>> chances but still no transformants. I am thinking that maybe the plasmid
>> concentration from the mini-prep is insufficient or too dirty for
>> electroporation? I am phenol:chloroform extracting the mini, however. I
>> was just wondering if anyone else has had the same difficulty in
>> rescuing their preys in KC8 and would greatly appreciate any suggestions
>> or advice!
>> Thanks so much,
>> Nancy
>>
>> nayers at cctr.umkc.edu
>> School of Biological Sciences
>> University of Missouri-Kansas City
>> 5007 Rockhill Road
>> Kansas City, MO 64110
>
>Hi!
>
>The strategy you describe works fine in my hands. I am also plating the
>transformation mix onto LB amp plates followed by replica plating onto
>selective medium. Plating directly onto M9 plates results in very low
>efficiency and the few colononies need about two day to become visible.
>I also made the experience that transformation efficiency differs
>between yeast plasmid preparations, although I always came up with
>at least some transformants. To me it seems that there is a little to
>little plasmid in your preparation if nothing at all. I would try to
>PCR your AD-insert using insert flanking primers this will show you
>whether your plasmid is present at all and it also tells you whehter
>you have a mixed population.
>What medium you use for growing the mini culture? You always have to
>keep pressure on both plasmids and select for interaction otherwise
>your plasmid might be lost if harmful for the cell.
>Hope this helps.
>
>Ricky
>
>******************************************************************
>
>Frederik Boernke
>Research Group of  Molecular Plant Physiology
>Institute for Plant Genetics and Crop Plant Research (IPK)
>Corrensstr. 3
>06466 Gatersleben
>Tel.  039482 -5 321
>Fax. 039482 -5 515
>http://www.ipk-gatersleben.de





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