Preparation of BAC DNA

BAC DNA Isolation with AutoGen 740

1. The BAC clone from glycerol culture sublibrary is innoculated into 3 ml of LB with 20µg/ml chloramphenicol and cultured at 37°C with shaking for 16hrs. The liquid bacterial culture is transferred into the tube specific for AutoGene 740 DNA Isolation System (Autogen, Inc.)

    

2. For BAC DNA purification, we use alkaline lysis approach by set up " System 1" in the panel of the AutoGen 740. Then, follow the manual provided by manufacturer.

    

3. Reagents

    

   • Reagent1: TE plus RNaseA (10 ml of Tris/HCl pH8.0; 2ml of 0.5M EDTA pH 8.0; 100mg RNaseA; Add ddH₂O to 1L. Prepared by ourself and stored at 4°C)
   • Reagent2: NaOH/Sarkcayl (0.2 N NaOH/1% N-Lauryl-Sarkosine. Prepared by ourself, and stored at room temperature)
   • Reagent3: KoAc/Phenol/chloroform/Ethanol (Ordered from Autogen, Cat No. AT000310) stored at -20°C
   • Reagent4: isopropanol
   • Reagent5: 70% Ethanol

   Make sure the reagent 1 and 3 are fresh for each run.

    

4. The program parameters have a few modifications as the following:

   Step No.	Process	Parameter set
   07	Agitation to resuspend cell pellet	80 Hz; 5 minutes
   19	Agitation to precipitate DNA	90 Hz; 5 minutes
   25	Centrifuge to pellet DNA	3500 rpm; 2 minutes
   33	Agitation to dissolve DNA	1Hz; 1 amp; 1 Second

    

5. Common yield of this approach is about 2µg BAC DNA/ 3ml Bacterial culture, but the quantity and quality of DNA are dependended on the BAC clone itself such as insert size etc. You can test DNA quality and quantity by digesting some of it with Hind III and running a 1% agarose gel for 2-3 hrs.