|
| |
|
|
| BAC DNA Isolation with AutoGen 740
|
| [ 文章来源： | 文章作者：
| 发布时间：2007-01-09|
字体：
[大
中
小]
| |
|
- 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.
上一篇：cDNA Production 下一篇：BENTON DAVIS BLOTS
|
|
|
cDNA Production