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I in the presence of RNaseH, made the ends blunt by treating with Klenow, Mung Bean nuclease, and then Klenow again (unpublished protocol developed by J. Gyuris), and ligated EcoRI adaptors onto them.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, lacI^qZ 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.

Center for Molecular Medicine & Genetics                

Wayne State University School of Medicine       

540 East Canfield Avenue                                          

Detroit, Michigan 48202

 http://proteome.wayne.edu/