Drosophila ovary acid fusion
library
(RFLY3) June
1993
This library is described in Finley
et al., 1996, PNAS 93, 3011-3015.
RNA
Wild type Drosophila
melanogaster ovary
RNA was isolated and poly(A)+ selected by Gerardo
Jimenez in David Ish-Horowicz's lab.
cDNA
I made unidirectional cDNA with an EcoRI sticky end at
the 5'-end and an XhoI sticky end at the 3'-end using a
method similar to the variation of the Gubler and
Hoffman technique (Gene 25: 263-269, 1983) described by
Huse and Hansen (Stratagene Strategies 1: 1-3, 1988). I
synthesized the first strand with Superscript (BRL)
using a primer (JG33) containing an XhoI site 5' to an
18nt poly(dT) tract. The first strand synthesis
contained 5me-dCTP instead of dCTP to protect internal
XhoI sites from subsequent cleavage with XhoI. I
synthesized the second strand with E. coli DNA pol I in the presence of
RNaseH, made the ends blunt by treating with Klenow,
Mung Bean nuclease, and then Klenow again (unpublished
protocol developed by J. Gyuris), and ligated EcoRI
adaptors onto them. The sequence of the adaptors is
shown below.
5'AATTCGGCACGAGGCG 3'
(JG31)
3'GCCGTGCTCCGC 5'
(JG32)
I cut the cDNA with XhoI and size
fractionated it using a 1 ml CL-4B column. The largest
fractions, representing cDNA greater than 0.3 kb, were
pooled and ligated into the unique EcoR1/XhoI sites of
plasmid pJG4-5 (ApR, TRP1+, yeast 2um
replicator) downstream of the GAL1 promoter, an ATG, and
sequences encoding the SV40 nuclear localization signal,
a hemagglutinin epitope tag, and the B42 acid patch
transcription activating domain (see pJG4-5
handout).
I transformed E.coli (Stratagene SURE cells: mcrA, L(mcrCB-hsdSMR-mrr)171,
endA1, supE44, thi-1, gyrA96, relA1, lac, recB, recJ,
sbcC, umuC::Tn5(Kanr), uvrC, [F', proAB, lacIqZ LM15, Tn10(Tetr)] ) with the
ligation mix and plated onto 21 LB amp plates (24cm X
24cm) and grew at 37oC for 20 hours.
I collected 3.5 X 106 independent
transformants
by scraping the plates (first cooled at 4oC) and thoroughly
resuspended the cells in one liter of LB amp. Aliquots
of 0.5 ml were combined with 0.5ml of 65% glycerol,
0.1M MgSO4, 25mM TRIS 7.4 and
stored at -70oC for future
amplification. The remainder was pelleted, frozen
overnight and used to prepare plasmid DNA (RFLY3) by two
CsCl gradients. DNA is at 0.6 mg/ml.
Over 87% of the plasmids of this library (RFLY3) contain
cDNA inserts whose sizes range from 0.3 to 1.5 kb
(average size about 800bp).
Russ
Finley Brent
lab - June 1993
Present Address:
Russell L. Finley
Jr.
Associate
Professor
Office (313) 577-7845
Center for Molecular Medicine
&
Genetics
Lab (313) 577-1627
3135
Scott
Hall
Fax (313) 577-5218
Wayne State University School of Medicine rfinley(at)wayne.edu
540
East Canfield
Avenue
http://cmmg.biosci.wayne.edu/
Detroit, Michigan
48202
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