We are encountering problems in constructing yeast genomic library. The
vector we use is pRS426 (a 2 micron-based plasmid) cut with BamHI and treated
with Calf Intestine Phosphatase. The yeast genomic DNA was purified by a
spooling method and partially digested with Sau3AI. DNA fragments from 6 to 12
Kb (or 2 to 6 Kb) were isolated from agarose gel and GeneCleaned. We have
tried to follow the advise by Rose and Broach (Methods in Enzymology, vol 194)
without a great deal of success. I would appreciate suggestions for the
following questions, or detailed protocol for constructing genomic libraries.
(1) What is the most appropriate means for preparing yeast genomic DNA? I
am concerned about the purity, potential inhibitors, intactness of the genomic
DNA. Is the spooling method acceptable for this purpose?
(2) What is the most suitable vector/insert ratio (by weight or by molar
ratio)?
(3) How many bacterial colonies one can expect to get from one small
scale, say 10 microliter, ligation reaction?
Any help will be greatly appreciated.
Tien-Hsien Chang
chang.108 at osu.edu
