BAC Related Information
Searching BAC clone by the BAC browser
- You can search BAC clones through BAC browser with gene symbols as a keyword, and confirm the physical location of BAC clones on the genome.
- Once you receive a BAC clone, inoculate the bacteria on an LB agar plate containing appropriate selective antibiotics within a few days after you received the plastic vial of stab culture.
- Pick up bacteria from the middle part of stab culture but not those from the surface of the stab which are not really healthy.
- CAUTION: The vial should be placed in a refrigerator (4°C) but never in a freezer, otherwise spoiled.
BAC DNA Preparation
- Ideally spread BAC on a LB plate to isolate single clolonies. Pick a single colony of BAC into 5mL of 2x LB culture containing a selective antibiotic – incubate at 37°C with shaking for 18-20 hrs.
- Collect the culture into a microtube, pellet the cells by centrifuge at 5,000 rpm for 5 mins in a bench top centrifuge, decant the supernatant.
- Resuspend the pellet in 300 uL of Qiagen solution P1 (cat no:19051) containing RNAaseA by pipetting – make sure that the cells are completely resuspended and no cell clumps!
- Buffer P1
50 mM Tris-Cl, pH 8.0; 10 mM EDTA; 100 ug/mL RNaseA
- Buffer P1
- Add 300 uL of Qiagen solution P2 (cat:19052), mix by hand-inversion 4 or 5 times (DO NOT VORTEX), incubate a room temp for 5 mins.
- Buffer P2
200 mM NaOH; 1% SDS (w/v)
- Buffer P2
- Add 300 uL of Qiagen solution P3 (cat 19053), seal the tubes and mix by inversion – leave on ice for 10 mins.
- Buffer P3
3 M potassium acetate, pH 5.5
- Buffer P3
- Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4°C.
- Transfer the entire supernatant to a fresh tube and precipitate the DNA by adding 700 uL of isopropanol, mix and leave at room temperature for 5 mins.
- Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4°C, carefully decant off the supernatant – white pellet should be visible. Wash with 70% EtOH then aspirate off ethanol. You do not need to remove all of the ethanol at this step, but you should minimize it.
- Add 50 uL of sterile TE (10mM Tris, pH 8, 1mM EDTA).
- Digest 8 uL of the DNA with 10 units of an appropriate restriction enzyme in 10 uL reaction mixture.
- Analyze the DNA by a pulsed-field gel electrophoresis.
Viability of recombinant E.coli in stab culture
- The viability of the E.coli were counted by flow cytometer at indicated days. (Okubo et al., unpublished)
Stab cultures of a recombinant E.coli, DH10B carrying a BAC clone of mouse MSM/Ms strain (MSMg01-174K01), were stored at different temperatures. Total and only dead cells were stained thiazole orange (TO) and propidium iodide (PI) , respectively.
Refer Protocol relating to the use of the deposited matelials in Lab Manual.
- Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones
- Sugimoto, M., Kondo, M., Hirose, M., Suzuki, M., Mekada, K., Abe, T., Kiyonari, H,. Ogura, A., Takagi, N., Artzt, K., Abe, K. Molecular identification of t(w5): Vps52 promotes pluripotential cell differentiation through cell-cell interactions. Cell Rep., 2 (5): 1363-1374 (2012). PMID: 23142660
- Yamaguchi, S., Niwa, R., Kazuki, Y., Ohbayashi, T. Application of a bacterial artificial chromosome modification system for a human artificial chromosome vector. Yonago Acta medica 54:021-031 (2011).
- Gong, S., Kus, L., Heintz, N. Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. Nat. Protoc., 5 (10), 1678-1696 (2010). PMID: 20885380
- Abe, K., Noguchi, H., Tagawa, K., Yuzuriha, M., Toyoda, A., Kojima, T., Ezawa, K., Saitou, N., Hattori, M., Sakaki, Y., Moriwaki, K., Shiroishi, T. Contribution of Asian mouse subspecies Mus musculus molossinus to genomic constitution of strain C57BL/6J, as defined by BAC-end sequence-SNP analysis. Genome Res., 14, 2439-2447 (2004). PMID: 15574823
List of articles regarding BAC clones
Please refer an article list in each BACs.
- B6N BAC [link]
- MSM/Ms BAC [link]
- Rat F344/Stm RNB1 BAC [link]
- Rat LE/Stm RNB2 BAC [link]
- Chinese hamster BAC [link]
- Drosophila melanogaster DME BAC [link]
- Drosophila simulans DSM BAC [link]
- Drosophila sechellia DSE BAC [link]
- Drosophila ananassae DNB BAC [link]
- Drosphila auraria DAB BAC [link]
(MAN0001e 2009.06.13 T.M.)