Drosophila (disc) acid fusion library
(RFLY5) June 1993
This library is described in
Finley et al., 1996, PNAS 93, 3011-3015.
RNA
Wild type Drosophila melanogaster disc RNA was
isolated by Janice Fisher
Vise in Ruth Lehman's lab. I selected
poly(A)+ RNA on an oligo (dT)
cellulose column and used it to
synthesize cDNA
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, see Jenö Gyuris handout) 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Bean nuclease, and then Klenow again (unpublished protocol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 0.5 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 19 LB amp plates (24cm X
24cm) and grew at 37oC
for
20 hours.
I
collected 4.0 X 107
independent
transformants by
scraping
the plates (first cooled at 4oC)
and thoroughly resuspended
the cells in one liter of LB amp. Aliqouts
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 (RFLY5)
Over 92% of the plasmids
of this library (RFLY5)
contain cDNA inserts whose sizes range from
0.3 to 2.1 kb (average
size about 900bp).
Amplification of the
above library June 1993
From
the original resuspended transformants, I diluted into 6
liters
LB-amp to OD600=0.1,
grew 24 hours at 37oC, and
stored the pellet at -20oC
until preparing plasmid
DNA.
Russ
Finley
Brent
lab - June 1993
Present Address:
Russell L. Finley
Jr.
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://proteome.wayne.edu/
Detroit, Michigan
48202
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