Preparation of BAC DNA

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.)

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.

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.

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

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.