| CHORI-230
Rat (BN/SsNHsd/MCW) BAC library
|
 |
The CHORI-230 BAC
Library has been constructed at the Children’s Hospital
Oakland Research Institute, BACPAC Resources by Drs Kazutoyo
Osoegawa and Baoli Zhu using the cloning techniques developed
in our laboratory (Osoegawa
et al., 1998). BAC library construction was funded by
a grant from NIH (HG01165-06). Rat brain genomic DNA was isolated
and partially digested with a combination of EcoRI and EcoRI
Methylase. Size selected DNA was cloned into the pTARBAC2.1
vector between the EcoRI sites. For segments 2 of the library,
agarose embedded high molecular weight DNA was partially digested
by MboI followed by double size fractionation on CHEF gel.
Size fractionated DNA was cloned into the pTARBAC1.3 vector
between the BamHI sites. The ligation products were transformed
into DH10B (T1 resistant) electrocompetent cells (BRL Life
Technologies). The library has been arrayed into 384-well
microtiter dishes and also gridded onto 22x22cm
nylon high-density filters for screening by probe hybridization.
Each hybridization membrane represents over 18,000 distinct
rat BAC clones, stamped in duplicate.
Rat Source: Howard
Jacob's laboratory (Medical College of Wisconsin). DNA was
isolated from a 3-month old female BN rat by Kazutoyo Osoegawa
during a visit to Dr. Howard Jacobs' lab in September 2000.
The complete strain nomenclature is BN/SsNHsd/MCW. BN stands
for Brown Norway. SsNHsd is a colony that was obtained from
a nucleus colony of Harlan Sprague Dawley (Hsd), at the NIH.
The rat BN/SsNHsd was brought to the Medical College of Wisconsin
(MCW) 17 generations ago. The rat used for construction of
the library is the 14th generation. Due to the incomplete
inbreeding of the original Hsd rats, Jacob's lab took care
of 17 additional generations of inbreeding towards more complete
homozygosity. A general idea of the strain, microsatellite
characterization and further links of interest can be obtained
from the following URL: http://rgd.mcw.edu
The CHORI-230
BAC Library:
| Segment |
|
DNA |
Restriction enzyme |
Plate Numbers |
Total Plates |
Empty Wells |
Empty Wells (%) |
| 1 |
pTARBAC2.1 |
Brain |
EcoRI |
1-240 |
240 |
1,742 |
1.9 |
| 2 |
pTARBAC1.3 |
Liver |
MboI |
241-528 |
288 |
1,677 |
1.5 |
| Total Library |
|
|
|
1-528 |
528 |
3,419 |
1.7 |
| Segment |
Non-insert Clones
(%) |
Non-insert clones
|
|
Recombinant Clones |
Average insert size |
Redundancy (Genome
size: 3,000 Mb) |
| 1 |
1.1 |
approx. 995 |
0 |
Approx. 89,423 |
214 Kbp |
6.4 |
| 2 |
6.8 |
7,406 |
2 |
Approx. 101,507 |
162 Kbp |
5.5 |
| Total Library |
4.2 |
8,401 |
2 |
Approx. 190,930 |
186 Kbp |
11.9 |
Data on the CHORI-230
clone average insert size has been determined by Pulsed
Field Gel Electrophoresis. Clone size distribution has been
plotted graphically. While analyzing clones using pulse-field
electrophoresis to determine the average insert size, non-insert
clones containing a small deleted vector fragment consistent
with sucrose resistance were observed. Further in depth characterization
of the library is on going in our lab and data will be updated
on our web page periodically.
The library is available
in several formats. Individual clones, and high-density hybridization
filters are obtainable. For ordering and shipping details,
please view the ordering
and pricing information page.
Academic and commercial
users interested in a copy of the BAC library should contact
Pieter J. de Jong ( pdejong@chori.org
), fax: (510) 450-7924).
Please direct questions
concerning this library to either Pieter
J. de Jong or Kazutoyo
Osoegawa .
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