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 E.coli 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.
- Please visit Recombineering Information in the Biological Resources Branch in the NCI-Frederick.
- BACPAC Resources Center, Children’s Hospital Oakland Research Institute
- 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
- 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).