|
CHORI-255: Gorilla (Male) (Gorilla gorilla gorilla) BAC Library |
 |
The CHORI-255 gorilla BAC library has been constructed by Michael Nefedov in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. The preparation of the library followed the cloning approach developed in our laboratory (Osoegawa et al., 1998). To obtain DNA from this endangered species, a proposal was submitted by Evan Eichler (Case Western Reserve University, Cleveland) to the Species Survival Plan Coordinator (SSCP). After favorable review, Dr. Lisa Faust (the SSCP) kindly provided us a gorilla blood sample. This gorilla was a male Western lowland gorilla (“Frank”, #327, Gorilla gorilla gorilla) housed at the Lincoln Park Zoo (Chicago). High-molecular-weight DNA was isolated from white blood cells by embedding in agarose. The embedded DNA was partially digested with a combination of EcoRI restriction enzyme and EcoRI Methylase and size fractionated by pulsed-field electrophoresis. DNA fragments from the appropriate size fraction were cloned into the pTARBAC2.1 vector between the two EcoRI sites. The ligation products were transformed into DH10B (T1 resistant) electro-competent cells (Invitrogen). The library has been arrayed into 576 “384-well” microtiter dishes and has subsequently been gridded onto twelve 22x22cm nylon high-density filters for probe hybridization screening. Each hybridization membrane represents over 18,000 distinct gorilla BAC clones (48 dishes), stamped in duplicate. Library characterization was performed by Qing Cao and Kazutoyo Osoegawa. BAC library construction was funded by NIH grants HG01165-07SI and HG025323-01 as part of the NIH-funded BAC Resource Network.
click here for a legend of the previous tables.
| Segment | 1 | 2 | Total | | Cloning Vector | pTARBAC2.1 | pTARBAC2.1 | | | DNA Source | White Blood Cell | White Blood Cell | | | Restriction Enzyme | EcoRI | EcoRI | | | Plate Range | 1-288 | 289-576 | 1-576 | | Total 384-Well Dishes | 288 | 288 | 576 | | Empty Wells (%) | 1,800 (1.6%) | 1,600 (1.4%) | 3400 (1.5%) | | Non-insert Clones (%) | 4/278 (1.4%) | 6/277 (2.1%) | 10/555 (1.8%) | | Vector Clones (%) | 2/110K (0) | 1/110K (0) | 3/220K (0) | | Recombinant Clones | >107K | >106K | >213K (>96% of wells) | | Insert size (Average) | ~200Kbp | ~170Kbp | ~185Kbp | | Standard Deviation | 39Kbp | 34Kbp | 36Kbp | | Library Redundancy | ~7 fold | ~6 fold | ~13 fold |
Data on the CHORI-255 clone average insert size has been determined by Pulsed Field Gel Electrophoresis. Clone size distribution has been plotted graphically.
Individual clones identified after screening can be obtained as stab cultures at a cost of $80 per clone in addition to a $35 set-up charge per shipment. Shipping will be done by Federal Express and charged to recipient's account number.
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]
Please contact Pieter J. de Jong for questions related to this library.
|
