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CHORI-236: Platypus (F) (Ornithorhynchus anatinus) BAC Library |
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The CHORI-236 “Platypus” BAC library has been constructed by Mikhail Nefedov 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 brain and spleen tissue obtained from a female platypus. This animal was obtained by Jenny Graves (Australian National University, Camberra, Australia) and is the same animal also used for whole genome sequencing at the Washington University Sequencing Center in St.Louis. The animal/tissue was collected by Frank Grutzner, Enkhjargal Tsend-Ayush (Australian National University, labratory of Jenny Graves) and Russell Jones (University of Newcastle). The agarose blocks were created by KeiJun Wei (Australian National University, Jenny Graves' lab). The agarose-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 864 384-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. 541 BAC clones from the first 541 dishes of the arrayed library have been analyzed via pulsed field gel electrophoresis of NotI-digested DNA. The results indicate that the average insert size is around 148 kb. The BAC library construction was funded by NIH grant (#HG025323-03) under the auspices of the NIH Resource Network.Segment | 1 | 2 | 3 | All | Vector | pBACGK1.1 | pBACGK1.1 | pBACGK1.1 | pBACGK1.1 | Restriction Enzyme | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | EcoRI/EcoRI Methylase | N/A | DNA Source | N/A | N/A | N/A | N/A | Plate Numbers | 1-288 | 289-576 | 577-864 | 1-864 | Plate Count | 288 | 288 | 288 | 864 | Empty Wells | 2477 (2.24%) | 2986 (2.7%) | 2395 (2.17%) | N/A | Non-Recombinant Clones | N/A | N/A | N/A | 44 (0.01%) | Non-Insert Clones | Approx. 818 (2/271, 0.74%) | Approx. 2013 (5/275, 1.82%) | N/A | Approx. 4247 (7/546, 1.28%) | Recombinant Clones | 107297 | 105593 | 108197 | 327485 | Average Insert Size | 137 Kbp | 157 Kbp | N/A | 147 Kbp | Genomic Coverage | N/A | N/A | N/A | N/A |
A total of No clones were found to be non-recombinant after analysis of CHORI-236 using overgo probes specific for the puc19 fragment.
Data on the CHORI-236 clone insert size distribution has been determined by pulsed-field gel Electrophoresis. Clone Size Distribution has been plotted graphically.
Hybridization filters and individual clones can be ordered online. Filters can be obtained only per complete set or per segment of the library (see Table for the plate number range for each of the segments). Shipping will be done by Federal Express and is preferably charged to the Fedex account number of the recipient. Information about cost and conditions are listed on our ordering instruction page. Web page updated: August 27, 2005
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