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constructed bacterial artificial chromosome
(BAC) library from TME3. A BAC library
was constructed in order to generate BAC con-
tigs around
CMD2
. The BAC library construc-
tion was carried out at the Clemson University
Genome Institute (CUGI) and the screening was
conducted at CIAT. Construction of the library
was as described by Tomkins and colleagues
(2004). To estimate the distribution and average
size of the clones, a total of 370 clones from the
TME3 library were picked randomly and grown
overnight in 3 ml of liquid LB medium
template for PCR amplification under standard
conditions.
The BAC library from TME3 has a 10.1
haploid genome equivalent, providing a 99%
chance of finding any particular sequence. The
BAC library was screened by PCR amplification
of 'BAC pools' (Moreno 2005). Results of the
'BAC pools' screening with NS158 yielded 2
positive clones, whereas screening with RME1
yielded 14 positive clones. NS158 is a single
copy SSR marker, whereas RME1 was devel-
oped from a multiple-copy RAPD marker. The
clones were digested with 20 U of
Hind
III
overnight and run for 24 hours on 1.2% agarose
gel to obtain a BAC-clone fingerprint (Figure
15.8). Positive clones were located using plate,
column, and row coordinates. The 16 positive
BAC clones were selected, fingerprinted, and
assembled into contigs associated with each
marker. The chromosome-walking approach to
the
CMD2
gene started with the contig con-
struction, using the FPC ('Fingerprinting Con-
tig') program (Marra et al. 1997), followed by
two methodologies (allele-specific primers and
SSCP-SNP) to design specific primers from the
BAC ends in order to develop new molecu-
lar markers closer to the
CMD2
gene region.
This process includes consecutive rounds of fine
mapping and BAC-library screening toward the
CMD2
gene.
The positive BAC clones were digested with
the
Hind
III enzyme and restriction profiles
were analyzed with the FPC program, using
an image.tif of the agarose gel. The astringent
parameters to define clone overlapping recom-
mended for the author were tolerance
+
12.5
μ
l chloramphenicol. Plasmid DNA was iso-
lated, digested with
Not
I restriction enzyme, and
inserts were separated from the vector by pulsed-
field electrophoresis.
A BAC library arrangement of plate, column,
and row pools was created, namely 'plate pools'
(PP), 'column plates' (CP), and 'row plates'
(RP). All 192 384-well plates were duplicated
using a 384-pin replicator and allowed to grow in
the LB/chloramphenicol (12.5
g/
μ
μ
g/ml) medium
at 37
◦
C overnight. For the BAC plate pool, all
the bacteria culture in a 384-well plate was com-
bined into an omnitray and 200
l of this trans-
ferred into a single well in a 96-well plate, to
yield 2 'BAC pool' plates. Simultaneously, every
ten plates of the library were inoculated into a
single 384-well plate using a 384-pin replica-
tor to give twenty 384-well plates. Each row of
each of the twenty plates was inoculated, using
a sterile toothpick, into a single well containing
the LB/chloramphenicol (12.5
μ
μ
g/ml) medium,
to form 'row plates (RP),' i.e., five RPs of 96-
well plates in total. The same was done for each
column of the twenty 384-well plates, combined
into a single well to form four 96-well 'column
plates (CP).' A total of eleven PPs, RPs, and
CPs was obtained. Contig construction was con-
ducted by PCR amplification of 'BAC pools.'
For PCR amplification, 5
=
7 and
1e
12
. The clones located at the ends
of each contig were identified and their BAC
ends were sequenced at the Iowa State University
facility sequencing center. The sequences were
edited for vector contamination and analyzed in
the genome database with the Blastx algorithm
(Altschul et al. 1997). Primers were designed
on each sequence, anticipating an amplification
product between 300-350 pb using the Primer
3.0 software (Rozen and Skaletsky 2000). The
cutoff
=
l of the bacteria cul-
ture was transferred, using a multi-pipette, to a
clean 96-well plate and the bacteria pelleted at
4,500 rpm for ten minutes in a Sorvall centrifuge.
The supernatant was discarded and the pellet re-
suspended in 5
μ
μ
l of sterile water and used as
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