Finley Lab
Center for Molecular Medicine and Genetics

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Center for Molecular Medicine and Genetics
Wayne State University School of Medicine
540 E. Canfield
Detroit, MI 48201


Drosophila (disc) acid fusion library (RFLY5)           June 1993


This library is described in Finley et al., 1996, PNAS 93, 3011-3015.



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



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I in the presence of RNaseH, made the ends blunt by treating with Klenow, Mung Bean nuclease, and then Klenow again (unpublished protocol developed by J. Gyuris), and ligated EcoRI adaptors onto them.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

540 East Canfield Avenue                                 

Detroit, Michigan 48202

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