Rat Sciatic Nerve library (RFSN) 2/17/97
2x106 independent clones, 80% with inserts
ranging from 0.5-2.5 kb
Plasmid DNA at 0.465 mg/ml in TE
E.coli primary transformants available (limited number of
aliquots).
Constructed by Wenbo Xu in the laboratory of Russ Finley, in
collaboration with Drs. John Kamholz and Michael Shy (Center for Molecular
Medicine and Genetics, Wayne State University School of Medicine, Detroit,
Michigan 48201).
The following description was taken from Wenbo Xu’s
Ph.D. Dissertation 2/2/00, with minor editing –RLF 7/15/00.
Rat sciatic nerve cDNA library construction.
(1)
Total RNA isolation and mRNA purification
A rat sciatic nerve cDNA library for expression of proteins
fused to a transcription activation domain in yeast was made using the 2
µm vector, pJG4-5 (Gyuris et al., 1993). Total RNA was derived from 10 rat sciatic nerves of 20-30
day old rats, the time of peak myelin gene expression. The total RNA was isolated using
guanidium-CsCl gradient centrifugation (Chirgwin et al., 1979). About 500
µg of total RNA was used to isolate poly(A)+ RNA using an oligo(dT)
cellulose column (Stratagene Cat # 200349). ~15-20 µg of poly(A)+ was recovered.
(2) cDNA synthesis
cDNA was synthesized unidirectionally, with an EcoRI sticky
end at the 5' end and an XhoI sticky end at the 3' end, using a variation of
the technique described by Gubler and Hoffman, 1983 (described in Finley et
al., PNAS 93, 3011-3015, 1996). 5
µg of poly(A)+ RNA was used to synthesize the first strand cDNA with
Superscript II reverse transcriptase (BRL) using primer JG33
(5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3') containing an
XhoI site (underlined) 5' to an 18 nucleotide poly(dT) tract. The first strand synthesis
contained me5-dCTP instead of dCTP to protect internal XhoI sites
from subsequent cleavage with XhoI.
The second strand was synthesized with DNA polymerase I (NEB) in the
presence of RNase H (Pharmacia). The
cDNA was then incubated with Pfu DNA polymerase (BRL) at 72oC for 30
minutes to make the ends double stranded
(blunt). The ends were
further trimmed by treating successively with Klenow (NEB), Mung Bean
Exonuclease (NEB), and Klenow. The
cDNA was purified using Sephacryl S-400 in a BMB G50 spin column, and then
ligated to EcoRI adaptors:
5'-AATTCGGCACGAGGCG-3'
3'-GCCGTGCTCCGC-5'
The cDNA was cut with XhoI and size fractionated using a 1
ml CL-4B column. The three largest
fractions, representing cDNA from ~0.5 kb to larger than 4 kb, were pooled,
treated with T4 kinase (NEB) and ligated to the unique EcoRI/XhoI sites of
plasmid pJG4-5.
(3) Transformation of E.coli with the rat sciatic nerve cDNA
The ligation mix was used to transform E.coli strain Sure (Stratagene). Cells were plated on 20 LB amp plates (24cmx24cm) and grown
overnight at 37oC. 2x106
independent transformants were collected by scraping the plates. Plasmid DNA was prepared using a Qiagen
kit after overnight growth in 2.5 liters of LB amp.
(4) Testing the library
Plasmid DNA was isolated from E.coli transformed with the cDNA library, and was cut with
EcoRI and XhoI and run on a 1.2% agarose gel. Over 90% of the plasmids had cDNA
inserts, with sizes ranging from 0.5-2.5 kb. PCR on 25 clones using primers for Po cDNA revealed that 1
in 25 was Po, consistent with the predicted level of Po expression at the peak
of myelination.
Contact:
Russ Finley
Center for Molecular Medicine
and Genetics
3135 Scott Hall
Wayne State University School
of Medicine
540 East Canfield Ave.
Detroit, Michigan 48201
Phone (313) 577-7845
e-mail rfinley(at)wayne.edu
or
Dr. John Kamholz
Center for Molecular Medicine
and Genetics
3101 Elliman
Wayne State University School
of Medicine
421 East Canfield Ave.
Detroit, Michigan 48201
Phone (313) 577-0925
e-mail jkamholz(at)genetics.wayne.edu
or
Dr. Mike Shy
Center for Molecular Medicine
and Genetics
3101 Elliman
Wayne State University School
of Medicine
421 East Canfield Ave.
Detroit, Michigan 48201
Phone (313) 577-6903
e-mail
mshy@genetics.wayne.edu
