Volume 17 Number 19 1989
Nucleic Acids Research
Rapid colony screening of YAC libraries by using aiginate as matrix support
Eric Lai* and Celeste Cantrell
Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599-7365, USA
Submitted August 25, 1989
The ability to clone large fragments of ENA in yeast artificial chromosome
(YAC) vectors^ has dramatically increased the ability to obtain physical naps
of complex genomes. Presently, transformation procedures that include a
spheroplasting step are preferred because of higher transformation efficiency.
The major obstacle in the preparation and screening of YAC libraries is the
large number of clones that must be manually picked from the top agar after
cell wall regeneration. To overcome this problem, aiginate has been used
instead of agar as the matrix in the top layer in constructing YAC libraries2.
Aiginate containing solution has the unique property of gelling in the
presence of calcium ions and reliquifing when the calcium is removed. We
report here a simple procedure using aiginate for spheroplast transformations
which allows complete transfer of YAC clones onto nylon membranes with very
little manual effort. A nylon membrane is placed on a 100 mm regeneration
plate containing 10 mM C a d 2 but lacking uracil. Transformed yeasts^ (100-200
fil) were mixed with 2 ml of 1.25% aiginate solution (FJC BioProducts,
Rockland, ME) and spread onto the membrane. The nylon membrane allows even
spreading of the aiginate mixture and provide the support for further
manipulation of the thin layer of aiginate gel. The aiginate will gal within
one hour and the plate is then kept at 30 *C for 24-36 hours. The aiginate gel
is then dissolved by transferring the nylon membrane with the top aiginate gel
onto a plate containing 1.5 % agar and 0.125 M EDTA. The agar will slowly
absorb the aiginate and the yeast colonies will be left on the nylon membrane.
The nylon membrane is then transferred to a plate which lacks uracil and
tryptophan. Replicates of the original colonies can then be made by plating
additional nylon membranes on the colonies.
We have found the same
transformation efficiency *4>ether using top agar, aiginate or according to
Ref.2. The main differences between our procedure and that of Ref.2 are the
use of the nylon membrane, the volume of aiginate used and removal of the
aiginate by EDTA. These modifications provide much more even spreading of
colonies and complete transfer of colonies onto the nylon membrane. Using this
procedure, 100,000 YAC clones can be transferred onto nylon membranes for
colony hybridization in two days with m-ln-limm manual
labor. Figure: Southern Blot hybridization of YAC
colonies on a replicate filter. Host strain AB1380 was
transformed with a YAC containing centromeric sequences
from chromosome I of S. pombe* QJ^ screened with a
single-copy probe.
AcknowleOmjuaiits: This work was
supported in part by a RMA Foundation Research Starter
Grant and a Medical Faculty Research Grant (UNC-CH).
E.L. is a TfiiVfiiTln Society of America Special Fellow.
We thank Drs. Clarke and Carbon for providing us with
the YAC clone pSp223 (cenl) 7L.
*To whom correspondence should be addressed
References 1. Burke et al., (1987) Science 22£, 806-812.
2. TTaver et a l . , (1989) Proc.Natl.Acad.Sci. fifi, 5898-5902.
3. Burgers and Percival, (1987) Anal. Biochem. 1£2, 391-397.
4. Hahnenberger et a l . , (1989) Proc.Natl.Acad.Sci. S&, 577-581.
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