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RPCI-13: Female Human BAC Library |
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The RPCI-13 Human Female BAC Library was constructed using improved cloning techniques developed by Kazutoyo Osoegawa. The library was generated by Baohui Zhao in our laboratory. Construction was funded by a grant from the National Human Genome Research Institute (NHGRI, NIH) (#1R01RG01165-03). The library was generated according to the new NHGRI/DOE "Guidance on Human Subjects in Large-Scale DNA Sequencing".
Female blood was obtained via a double-blind selection protocol. Female blood DNA was isolated form one randomly chosen donor (out
of 10 female donors) and partially digested with a combination
of EcoRI and EcoRI Methylase for library segments 1&2
or either MboI or DpnII for library segments 3&4.
Size selected DNA was cloned into the pBACe3.6 vector between
the EcoRI sites for library segments 1&2 or the BamHI
sites for library segments 3&4. 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-13 Female
Human BAC Library :
| Segment |
Cloning Vector |
Cloning Enzyme |
DNA |
Plate Numbers |
Total Plates |
Total Clones |
Empty Wells (Total) |
| 1 |
pBACe3.6 |
EcoRI |
Female |
1-240 |
240 |
89,834 |
2,326 |
| 2 |
pBACe3.6 |
EcoRI |
Female |
241-480 |
240 |
89,920 |
2,240 |
| 3 |
pBACe3.6 |
Mbol/Dpnll |
Female |
481-768 |
288 |
109,365 |
1,227 |
| 4 |
pBACe3.6 |
Dpnll |
Female |
769-1056 |
288 |
109,471 |
1,121 |
| Total Library |
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|
|
1-1056 |
1056 |
398,590 |
6,914 |
Segment |
Empty Wells (%) |
Non-Recombinant Clones (Total) |
Non-Recombinant Clones (%) |
Average Insert Size |
Genomic Coverage |
| 1 |
2.5 |
approx. 550 |
0.6 |
182 Kbp |
5.4X |
| 2 |
2.4 |
approx. 1,150 |
1.3 |
192 Kbp |
5.7X |
| 3 |
1.1 |
approx. 1,550 |
1.4 |
149 Kbp |
5.3X |
| 4 |
1.1 |
approx. 660 |
0.6 |
149 Kbp |
5.4X |
| Total Library |
1.7 |
approx. 3,910 |
1.0 |
166 Kbp |
21.8X |
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 form plates from each segment.
The library is available as library plates, individual clones, and high-density
hybridization sets (see Filter Availability)
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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