西亞試劑：：BAC DNA Isolation with AutoGen 740

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.

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