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BAC Related Information

BAC Related Information

  • When you receive the plastic vial of stab culture, please streak the bacteria on an LB agar plate containing a selective antibiotic. We hope you can find colonies.
  • When you streak bacteria, please take bacteria from the middle part of stab agar medium but not those from the surface of the stab which are not really healthy.
  • The vial should be placed in a refrigerator (4 C degree) but not in a freezer. Stab culture is not meant to be for frozen storage of bacteria.

BAC DNA Preparation

  1. 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 degrees centigrade with shaking for 18-20 hrs.
  2. 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.
  3. 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
  4. 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)
  5. 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
  6. Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4 degrees centigrade.
  7. 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.
  8. Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4 degrees centigrade, 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.
  9. Add 50 uL of sterile TE (10mM Tris, pH 8, 1mM EDTA).
  10. Digest 8 uL of the DNA with 10 units of an appropriate restriction enzyme in 10 uL reaction mixture.
  11. Analyze the DNA by a pulsed-field gel electrophoresis.

Related Information

Viability of recombinant E.coli in stab culture

  • 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.
    The viability of the E.coli were counted by flow cytometer at indicated days. (Okubo et al., unpublished)

Recombineering Information

Related Website

Related Articles

  • 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

(T.M. 2009.06.13)