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 Home > Resources > Libraries
   VMRC-26: Stickleback fish (Gasterosteus aculeatus) BAC Library   

The 3-spined stickleback (Gasterosteus aculeatus) BAC library was constructed by Andrew Stuart in Chris Amemiya’s laboratory at the Benaroya Research Institute at Virginia Mason (BRI) in support of the stickleback genome project. This library was designated VMRC-26. Blood cells were obtained from a single female specimen (#317-2 from Bear Paw Lake in Alaska) that was shown to exhibit the lowest levels of heterozygosity among 16 specimens surveyed at the Broad Institute. Cells were embedded in Incert agarose and high molecular weight DNA was prepared in situ in the agarose. Generation of the library closely followed the cloning approach developed in Pieter de Jong’s laboratory Osoegawa et al., 1998. Partial-digest EcoRI fragments from the appropriate size fraction were cloned into the pCCBACE1 vector (Epicentre Technologies). The ligation products were then transformed into DH10B (T1 phage resistant) electro-competent cells (Invitrogen). The library has been arrayed into 250 384-well microtiter dishes and has subsequently been gridded onto 22 x 22 cm 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. Analysis of 100 randomly selected BACs via pulsed field gel electrophoresis of NotI-digested DNA indicated that the average insert size was around 165 kb. The BAC library construction was funded by NIH grant HG02526 (Chris T. Amemiya, BRI) under the auspices of the NIH Resource Network.
Please note: This library has been used in support of the Stickleback genome project. In the process, many clones have been BAC-end-sequenced. The BAC-end-Sequences (BES) [190,040] can be found in the NCBI Trace Archive using the following query string: center_project="G821"

Segment

1

All

Vector

pCC1BAC

pCC1BAC

Restriction Enzyme

EcoRI

EcoRI

DNA Source

blood

blood

Plate Numbers

1-250

1-250

Plate Count

250

250

Empty Wells

N/A

N/A

Non-Recombinant Clones

4800 (5%)

4800 (5%)

Non-Insert Clones

N/A

N/A

Recombinant Clones

91200

91200

Average Insert Size

165 Kbp

165 Kbp

Genomic Coverage

20X

20X

 

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