RPCI - 13 Female Human BAC Library
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 | 89,284 | 2,326 |
| 2 | pBACe3.6 | EcoRI | Female | 241-480 | 240 | 88,770 | 2,240 |
| 3 | pBACe3.6 | Mbol/Dpnll | Female | 481-768 | 288 | 107,815 | 1,227 |
| 4 | pBACe3.6 | Dpnll | Female | 769-1056 | 288 | 108,811 | 1,121 |
| Total Library | 1-1056 | 1056 | 394,680 | 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.