Minipreparation of BAC DNA

Media and reagents

- Chloramphénicol: 12,5 mg/ml in ethanol 100

stock solution stored at -20C

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 37C 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 20C 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.