|
CHORI-235: Little Brown Bat (Myotis lucifugus) BAC Library |
 |
The CHORI-235 “Little Brown Bat” BAC library has been constructed by Boudewijn ten Hallers in Pieter de Jong’s laboratory at BACPAC Resources, Children’s Hospital Oakland Research Institute. The preparation of the library followed the general cloning approach developed in our laboratory (Osoegawa et al., 1998). DNA was isolated from frozen kidney tissue obtained from a female bat, identified by Utah specimen number “W31-04”. This animal was collected in Wyoming during the spring of 2004 by Russell Ray, Department of Human Genetics, University of Utah, Salt Lake City, Utah. High-molecular-weight DNA was isolated by creating a chromatin suspension from powder derived from frozen kidney tissue treated under liquid nitrogen using a pestle and mortar. The suspension was further homogenized using a Wheaton Dounce Homogenizer (7ml homogenizer with a tight pestle with 0.0010"-0.0030" clearance). The chromatin particles were embedded in 0.5% agarose gel plugs prior to deproteinizing the DNA. 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 pBACGK1.1 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 384-well microtiter dishes and has subsequently been gridded onto 22x22cm nylon high-density colony filters for screening by probe hybridization. Each hybridization membrane represents over 18,000 distinct BAC clones (equivalent to 48 "384-well" dishes), stamped in duplicate. The BAC library construction was funded by NIH grant (#HG025323-03) under the auspices of the NIH Resource Network.
Segment | 1 | 2 | All | Vector | pBACGK1.1 | pBACGK1.1 | pBACGK1.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | DNA Source | kidney | kidney | kidney | Plate Numbers | 1-288 | 289-528 | 1-528 | Plate Count | 288 | 240 | 528 | Empty Wells | 3815 (3.45%) | 2673 (2.9%) | N/A | Non-Recombinant Clones | 1 (0%) | 2 (0%) | 3 (0%) | Non-Insert Clones | Approx. 509 (1/216, 0.46%) | Approx. 1152 (3/240, 1.25%) | Approx. 1784 (4/456, 0.88%) | Recombinant Clones | 106267 | 88333 | 200965 | Average Insert Size | 176 Kbp | 157 Kbp | 161 Kbp | Genomic Coverage | 6.2X | 4.6X | 10.8X |
click here for a legend of the previous tables.
No clones were found to be non-recombinant after analysis of CHORI-235 using overgo probes specific for the puc19 fragment.
The library is available for individual clone orders distributed as agar-stab cultures. Clones can be identified through screening of "high-density colony" hybridization filters. The library may also be available to academic users by reimbursement of duplication costs (labor & materials). Dishes can only be obtained per complete library or per segment (about half of the library). Shipping will be done by Federal Express and charged to the recipients 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 questions about hybridization membranes, please contact BACPAC Resources ([email protected]).
Posted: October 16, 2004.
Revised: October 9, 2008
|
