Useful All Purpose Yeast:coli Vectors

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri May 7 19:09:00 EST 1993


Hello fellow Yeast netters, 
    I would like to inform all of you out there
who don't know about the YXplac vectors what you 
are missing.  In 1986, being a new yeast convert, I got
very tired of fooling around trying to clone things into
the standard vectors like YCp50, YEp24, YCp19 etc.  As you 
all know these vectors are full of restriction sites making
it very difficult to use with any type of fancy cloning.  I decided to 
make a set of yeast E.coli shuttle vectors using pUC19, at that
time and still my favourite coli cloning vector. (Many Thanks to 
J. Messing and crew!!!)  At the time there were a number of
other yeast E.coli shuttle vectors around (pEMBLY Baldari &Cesareni
Gene35:27-32[1985] as well as those made by Hill et al
Yeast 2:163-167 [1986]) but there was still the
problem of restriction sites within the yeast genes that made
it hard to work with the an EcoRI fragment when dealing with 
the LEU2 gene.  Working down the hall from Dr. Tom Kunkel at 
NIEHS I decided (with his expert advice and some help) to remove
 all the restrictions sites that were
found in the pUC19 multicloning site from all yeast DNA sequence
either by dut ung mutagenesis or blunt end filling.  
I removed the EcoRI and KpnI sites from the LEU2 gene
as well as the HindIII, XbaI and PstI sites from TRP1 ARS1 frag
and the Pst1 and SmaI (an easy one) sites from the URA3 fragment.
    With these Genes I built YCplac, YEplac and YIplac vectors
    for each selectable marker.

SEE Gietz and Sugino (1988) Gene 74:527-534

  Selectable Marker    TRP1       URA3        LEU2

ARS1 CEN4 plasmid    YCplac22    YCplac33    YCplac111
2 micron plasmid     YEplac112   YEplac195   YEplac181
Integrating plasmid  YIplac204   YIplac211   YIplac128

Advantages of these plasmids include small size (max 6.11kb),
Blue:white color selection for inserts
as well as the ability to use all the M13 primer for sequencing directly.
Each ofthe restriction sites in the multicloning site are unique.
This is a real
benefit for those doing alot of cloning, subcloning as well
as sequencing. Another benefit (as with most of pUC based yeast coli vectors) 
is the very good plasmid yield from 2 mls of miniprep DNA. 
I do a 2ml mini and have enough quality DNA for about 3-4 sequencing Reaxns.
I am giving these vectors to all who need them for research purposes.
I only have two conditions 1) you reference Gietz and Sugino properly
in any publication were they are used and 2) that you refer all those wanting
these vectors to me so I may keep track of who are using them.
I have also compiled DNA sequence file for each vector made by piecing 
together all the fragments. They too are available for research purposes.
If you feel that you can make use of these vectors by all means drop
me a line with your snail mail address and I will send them out to you.

______________________________________
R.Daniel Gietz Ph.D. (Dan)
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)788-6458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
________________________________________




More information about the Yeast mailing list