Public EST Project for Soybean
Record of Deposit
Soybean cDNA Library Gm-c1027
Degenerating Cotyledon library
The soybean cDNA library is being made publicly available by the following laboratory. To insure
that credit is given to the laboratory and scientists donating this valuable resource to the scientific
community, the information provided with the library must be maintained and include with any
transfer of biological materials or data derived from the library.
Source of Library:
Dr. Paul Keim
Virginia H. Coryell
Dept of Biology
Box 5640
Northern Arizona University
Flagstaff, AZ 86011
ph: 520-523-1078, 520-523-1372
FAX: 520-523-0639
e-mail: paul.keim@nau.edu
virginia.coryell@nau.edu
Short Description of Librarv:
The mRNA was isolated from cotyledons of 3- and 7-day-old ‘Williams’
seedlings which were propagated on paper towels with distilled water. The
cotyledons were flash-frozen in liquid nitrogen, then lyophilized for 72 hours.
Unequal amounts of mRNA were isolated, however all the mRNA was used for
cDNA synthesis.
Stratagene’s cDNA Synthesis Kit (catalog number 200401) was used to
synthesize the cDNA. First-strand synthesis was performed with 5-methyl dCTP,
hence the ligated cDNA was hemimethylated. A modification of Stratagene’s firststrand synthesis primer was used. An “anchor” nucleotide (V=A, C, or G) was
added to the 3’ end of the primer
[GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG(T)18V] to anchor the primer
at the 5’ end of the poly(A) tract. After second-strand synthesis, the cDNA ends
were filled in with cloned Pfu DNA, ligated to EcoRI adapters and subsequently
phosphorylated. The XhoI site within the first-strand synthesis primer was then
restricted by digestion with XhoI; all XhoI sites in the cDNA would be protected
by their hemimethylated status. The cDNA constructs were size-fractionated with
a 500 bp cutoff, using GibcoBRL Life Technologies’ cDNA Size Fractionation
column. The column eluent was then ligated into Stratagene’s pBluescript  II XR
Predigested vector (pBluescript II SK(+) that has been digested with EcoRI and
XhoI, and phosphorylated by Stratagene). 97% of the white and blue colonies
appear to contain recombinant plasmids with cDNA inserts, based on size (n=30)
Genotype/Cultivar: Williams
Vector:
pBluescript II XR
Other Information:
575 ul of transformed DH10B, approximately 240,350 cfu, in SOB and 8% glycerol have
been submitted.
ligation efficiency [(white cfu/total cfu)x100] = 59.1%
transformation efficiency = 1.66108 cfu/ug total DNA (GibcoBRL ElectroMAX DH10B
cells)
average insert sizes:
white colonies = 1044 bp (range from 0 to 2400 bp), n=20
blue colonies = 520 bp (range from 350 to 650 bp), n=5
overall insert average size = 830 bp
Expected vector sequence flanking root nodule CDNA clones available through Genome Systems
or contributing investigator.
Expected vector sequence flanking degenerating cotyledon cDNA library
clones:
826 M13 Reverse primer
T3 primer
5’GGAAAC AGCTATGACC ATG 3’
5’A ATTAACCCTC ACTAAAGGG 3’
5’GGAAAC AGCTATGACC ATGATTACGC CAAGCGCGCA ATTAACCCTC
ACTAAAGGGA
3’CCTTTG TCGATACTGG TACTAATGCG GTTCGCGCGT TAATTGGGAG
TGATTTCCCT
SK primer
5’CGCTCTAGA ACTAGTGGAT C 3’
ACAAAAGCTG GAGCTCCACC GCGGTGGCGG CCGCTCTAGA ACTAGTGGAT
CCCCCGGGCT
TGTTTTCGAC CTCGAGGTGG CGCCACCGCC GGCGAGATCT TGATCACCTA
GGGGGCCCGA
EcoR I adapter
Xho I site
5’AATTC GGCACGAG 3’
5’CTC GAG 3’
GCAGGAATTC GGCACGAG---cDNA---(A)nCTC GAGGGGGGGC CCGGTACCCA
CGTCCTTAAG CCGTGCTC---cDNA---(T)nGAG CTCCCCCCCG GGCCATGGGT
3’G CCGTGCTC 5’
EcoR I adapter
ATTCGCCCTA TAGTGAGTCG TATTACGCGC GCTCACTGGC CGTCGTTTTA C 3’
TAAGCGGGAT ATCACTCAGC ATAATGCGCG CGAGTGACCG GCAGCAAAAT G 5’
3’CGGGAT ATCACTCAGC ATAATG 5’ 3’TGACCG GCAGCAAAAT G 5’
T7 primer
M13 –20 primer 600