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
- Ideally spread BAC on a LB plate to isolate single clolonies. Pick a single colony of BAC into 2ml of 2x LB culture containing a selective antibiotic - incubate at 37 degrees centigrade 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!
50 mM Tris-Cl, pH 8.0; 10 mM EDTA; 100 ug/ml RNaseA
- 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.
200 mM NaOH; 1% SDS (w/v)
- Add 300 ul of Qiagen solution P3 (cat 19053), seal the tubes and mix by inversion - leave on ice for 10 mins.
3 M potassium acetate, pH 5.5
- Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4 degrees centigrade.
- 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 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.
- Add 50 ul of sterile TE (10mM Tris, pH 8, 1mM EDTA).
- Digest 5 ul of the DNA with 10 units of an appropriate restriction enzyme in 20 ul reaction mixture.
- Analyze the DNA by a pulsed-field gel electrophoresis.
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)
- Please visit
Recombineering Information in the Biological Resources Branch in the NCI-Frederick.
- BACPAC Resources Center, Children's Hospital Oakland Research Institute
- 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:
- 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