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 Home > Resources > Libraries
   VMRC-18: Opossum (F) (Monodelphis domestica) BAC Library   

  Background information

The laboratory opossum (Monodelphis domestica) BAC library was constructed by Andrew Stuart in Chris Amemiya’s laboratory at the Benaroya Research Institute at Virginia Mason. This library was designated VMRC-18. Brain tissue from a female laboratory opossum was provided by Paul Samollow, Southwest Foundation for Biomedical Research, San Antonio, Texas. This animal (San Antonio ID# I-1438) exhibited the lowest average heterozygosity (~1 heterozygous base per 4,200 bp) among numerous inbred animals examined. The animal for this effort was kindly provided by John VandeBerg from his breeding colony. DNA from this animal was used to generate whole-genome sequence at the Broad Institute Genome Sequencing Center, and this BAC library serves as a linking tool to better assemble the sequence. For BAC library construction, nuclei were isolated from the brain tissue using a Dounce homogenizer and sequential centrifugation steps. Nuclei were then 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). . DNA fragments from the appropriate size fraction were cloned into the pCC1BAC vector (Epicentre Technologies). The ligation products were then 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. Analysis of 120 randomly selected BACs via pulsed field gel electrophoresis of NotI-digested DNA indicated that the average insert size was around 175 kb. The BAC library construction was funded by NIH grant HG02526-01 (Chris T. Amemiya, BRI) under the auspices of the NIH Resource Network.
Please note: The following data is provisional pending further characterization.

Segment

1

2

All

Vector

pCC1BAC

pCC1BAC

Restriction Enzyme

EcoRI/EcoRI Methylase

EcoRI/EcoRI Methylase

N/A

DNA Source

brain

brain

N/A

Plate Numbers

1-528

529-1000

1-1000

Plate Count

528

472

1000

Empty Wells

N/A

N/A

0 (0%)

Non-Recombinant Clones

N/A

N/A

N/A

Non-Insert Clones

Approx. 10138 (5%)

Approx. 9062 (5%)

Approx. 19200 (5%)

Recombinant Clones

192614

172186

364800

Average Insert Size

175 Kbp

175 Kbp

175 Kbp

Genomic Coverage

7.9X

7.1X

15X


click here for a legend of the previous tables.

The library is available for individual clone orders distributed as agar-stab cultures at a cost of $80 per clone in addition to a $35 set-up charge per shipment. Clones can be identified through screening of "high-density colony" hybridization filters . 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.

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 (pdejong@chori.org, fax: (510) 450-7924). For questions about hybridization membranes, please contact BACPAC Resources (BACPACorders@chori.org).

Posted: November 30, 2004.
Revised:

 

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