Gm-c1078
Public EST Project for Soybean
Record of Deposit
Soybean cDNA Library Gm-c1078
48-hour Infected Supernod Root 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 roots of 7-day-old ‘Bragg’ supernodulating mutant
NTS382 seedlings that were infected with Bradyrhizobium japonicum, strain USDA 110,
48 hours prior to harvest. Dr. Gary Stacey generously donated the tissue. The roots were
flash-frozen in liquid nitrogen.
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 first-strand
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 cDNA was then precipitated and redissolved in sterile,
RNase-, DNase-free water. The XhoI site within the first-strand synthesis primer was then
restricted by digestion with XhoI from Promega (40U/ul); all XhoI sites in the cDNA
would be protected by their hemimethylated status. The cDNA constructs were sizefractionated with a 500 bp cutoff, using Sephacryl S-500 High Resolution (Pharmacia
Biotech) in a 2-mm diameter column and a bed volume of approximately 1.3 mL. The
column eluent was precipitated, redissolved, and ligated into Stratagene’s pBluescript II
XR Predigested vector (pBluescript II SK(+) vector that has been digested with EcoRI
and XhoI, and phosphorylated by Stratagene).
Genotype/Cultivar: ‘Bragg’ supernodulating mutant NTS382
Vector: pBluescript II SK(+)
Other Information:
~1200 ul of transformed DH10B cells, approximately 20,890 cfu, in SOB and 8% glycerol,
have been submitted. More library is available for further transformations.
ligation efficiency [(white cfu/total cfu)x100] = 80.4%
average insert sizes:
white colonies = 723 bp (range from 400 to 1200 bp), n=11
Expected vector sequence flanking 48-hour Infected Supernod Root cDNA clones available
through Genome Systems or contributing investigator.
Expected vector sequence flanking 48-hour Infected Supernod Root 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