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RPCI-11: Human Male BAC Library |
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The RPCI-11 Human Male BAC Library
(Osoegawa et al., 2001) was constructed
using improved cloning techniques (Osoegawa
et al., 1998) developed by Kazutoyo
Osoegawa. The library was generated by Kazutoyo
Osoegawa. Construction was funded by a grant from the
National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165-03).
This library was generated according to the new NHGRI/DOE
"Guidance on Human Subjects in Large-Scale DNA Sequencing".
Male blood was obtained via a double-blind selection protocol. Male blood DNA
was isolated from one randomly chosen donor (out of 10 male
donors) and partially digested with a combination of EcoRI
and EcoRI Methylase. Size selected DNA was cloned into
the pBACe3.6 vector between the
EcoRI sites. For Segment 5, the same male donor DNA was
partially digested with MboI, size selected, and ligated into
the pTARBAC1 cloning vector at the BamHI sites. The ligation
products were transformed into DH10B 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.
The RPCI Human Male BAC Library:
Segment |
Cloning
Vector |
DNA |
Plate Numbers |
Total Plates |
Total Clones |
Empty Wells (Total) |
1 |
pBACe3.6 (1) |
Male |
1-288 |
288 |
108,499 |
2,093 |
2 |
pBACe3.6 (1) |
Male |
289-576 |
288 |
109,496 |
1,096 |
3 |
pBACe3.6 (1) |
Male |
577-864 |
288 |
109,657 |
935 |
4 |
pBACe3.6 (1) |
Male |
865-1152 |
288 |
109,382 |
1,210 |
5 |
pTARBAC1 (2) |
Male |
1153-1440 |
288 |
106,763 |
3,289 |
Total Library |
|
|
1-1440 |
1440 |
543,797 |
9,163 |
1:
donor DNA EcoRI partially digested
2: donor DNA MboI partially digested
| Segment |
Empty Wells (%) |
Non-Recombinant
Clones (Total) | Non-Recombinant Clones (%) |
Insert
Size (average) |
Genomic
Coverage |
| 1 |
1.9 |
approx. 1800 |
1.7 |
164 Kbp |
5.8X |
| 2 |
1.0 |
approx. 550 |
0.5 |
168 Kbp |
6.0X |
| 3 |
0.8 |
approx. 1100 |
1.0 |
181Kbp |
6.7X |
| 4 |
1.1 |
approx. 1100 |
1.0 |
183Kbp |
6.8X |
| 5 |
3.5 |
approx. 530 |
0.5 |
196Kbp |
6.9X |
| Total Library |
1.7 |
approx. 5080 |
0.9 |
178 Kbp |
32.2X |
click here
for a legend of the previous tables.
The average
insert size has been determined by Pulsed Field Gel
Electrophoresis analysis of clones randomly chosen from plates from each segment.
Reference:
1.
Osoegawa, K., Mammoser, A. G., Wu, C., Frengen, E., Zeng,
C., Catanese, J. J., de Jong, P. J. (2001) A Bacterial Artificial
Chromosome Library for Sequencing the Complete Human Genome,
Genome Research, Vol. 11, Issue 3, 483-496, March 2001
(Reprints available upon request)
2.
Osoegawa, K., Woon, P.Y., Zhao, B., Frengen, E., Tateno, M.,
Catanese, J.J, and de Jong, P.J. (1998) An Improved Approach
for Construction of Bacterial Artificial Chromosome Libraries,
Genomics 52, 1-8; Article # GE985423. (Reprints available
upon request)
The library is available for individual
clone orders, distributed as agar-stab cultures at a not-for
profit cost. See our webpage "Ordering
& Pricing Information" for the costs .
The BAC library has been gridded onto 22x22cm positively charged
nylon filters for hybridization screening purposes. Each filter
contains 36,864 colonies which represents 18,432 independent
clones spotted in duplicate in a 4x4 clone array. Clones can
be identified through screening by purchasing "high-density
colony" hybridization filters or by utilizing our "fee
for service" "Library Screening
Services" . For questions about clones and hybridization
membranes, please contact BACPAC Resources ([email protected]).
With respect to the scheduling of shipments of arrayed library copies, we would
like to make you aware of our policy for array distribution.
Please visit the ordering information
page if you want to place an order.
Academic and commercial users interested in a copy of the BAC library should
contact Pieter J. de Jong ([email protected], fax: (510) 450-7924). For hybridization
membranes, please contact BACPAC Resources ([email protected]).
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