BAC Related Information

BAC DNA Preparation

1. 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.
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 5 ul of the DNA with 10 units of an appropriate restriction enzyme in 20 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 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.

Recombineering Information

• Please visit Recombineering Information in the Biological Resources Branch in the NCI-Frederick.
  

Related Website

• BACPAC Resources Center, Children's Hospital Oakland Research Institute
  http://bacpac.chori.org/protocols.htm

Related Articles

• 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).