Minipreparation of BAC DNA
Media and reagents
- Chloramphénicol: 12,5 mg/ml in ethanol 100
stock solution stored at -20°C
Dilute 1:1000 in LB medium
- LB medium + 12,5
mg/ml chloramphénicol- RNase A
- Cold GTE Buffer: glucose 50 mM, Tris-HCl 25 mM, EDTA 20 mM pH8
- Glucose 2M 250
ml- EDTA 0,5M pH8 400
ml- Tris-HCl 1M, pH8 250
ml--------
- H2O qsp ................. 10 ml
- NaOH-SDS: NaOH 0,2N et SDS 1%
- NaOH 10N 100
ml- SDS 10% 500
ml--------
- H2O qsp .............. 5 ml
- Potassium acetate 1,32M Kac 19,5 g
Acetic acid 10,5 ml
-------
H2O qsp .............. 150 ml
The final pH is 4,8
Culture
Inoculate a single colony in 3 ml LB + chloramphenicol
Let the bacteria grow at 37°C for 16h with vigorous shaking (250 rpm)
Purification of DNA
Transfer the cultures in 1.5 o 2 ml microtubes and centrifuge 1 min à 13.000 rpm or 5 min à 5000 rpm. Discard the supernatants
Resuspend the pellets in 200 ml cold GTE buffer + 100mg/ml RNaseA
Add 200 ml NaOH-SDS, mix by inverting the tubes twice and let at room temperature no more than 2 min
Add 200 ml cold potassium acetate, mix by inverting the tubes and let 5 min on ice
Centrifuge 15 min at maximum speed and carefully transfer the supernatants into 400 ml isopropanol. Mix by inverting the tubes.
Centrifuge 20 min at maximum speed. Discard the supernatants
Resuspend pellets in 100 ml TE and precipitate again by adding 10 ml Sodium acetate 3M and 250 ml ethanol
Let the tubes at –20°C for 1h
Centrifuge at maximum speed for 20 min and rinse the pellets with 70% ethanol
Dry the pellets
Add 30 to 50 ml TE 10.1 pH 8
Quantify DNA by loading 1 ml of the DNA solution on a 1% agarose minigel.