BAC DNA Midiprep Protocol

Reagents

Solution I ; 50 mM glucose, 10 mM EDTA, 25 mM Tris-Cl, pH 8.0

(for 100 ml) 5 ml of 1M glucose, 2.5 ml of 0.5M EDTA, 2.5 ml of 1M Tris and 90.5 ml water

Solution II ; 0.2 N NaOH, 1 % SDS

(for 100 ml) 0.8 g NaOH and 1 g SDS in 100 ml of water

Solution III ; 60 ml 5 M potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml dd H2O

(for 60 ml of 5 M potassium acetate) 29.45 g in 60 ml of water

All solution could be stored at room temperature.

QIAEX II kit

Every step including pipetting should be gentle and slow to avoid shearing the big DNA molecules.

A. Midi-prep Isolation of BAC DNA

Prepare a 100 ml culture of LB medium containing 12.5 ug/ml of chloramphenicol

Inoculate half loop (1 cm diameter) of cells and incubate with shaking at 37 degree for 18 20 h

Centrifuge the overnight culture by centrifugation at 5000 g for 15 min.

Pour off the supernatant fluid and resuspend the cell pellet in 4 ml of Sol I.

Add 8 ml of Sol II and invert gently. Incubate the solution at RT for 2 min and then the solution should turn translucent.

Add 6 ml of the Sol III and invert gently. A white precipitate should form. Keep the solution on ice for more than 30 min.

Centrifuge at 5000 g for 20 min to collect the precipitate.

Carefully transfer the supernatant to a fresh tube. (Use cheese cloth to prevent the white precipitate from coming up with supernatant)

The above solution could be kept at 20 degree for a few month. If you need crude BAC DNA, go to step 10.

Take 750 ul of the supernatant in step 8 and transfer to a clean microcentrifuge.

Add 450 l of isopropanol and gently mix. Centrifuge for 15 min at top speed (about 15000 g).

Remove the supernatant fluid and rinse the pellet with 1 ml of ice cold 70 % EtOH. Centrifuge for 5 min and pour off the EtOH rinse.

Air dry the pellet but do not overdry.

Resuspend the pellet with 100 ul of H2O + RNAse 50 ug/ml and incubate at 37 degree for 60 min.

B. Further purification of BAC DNA

Add 3 vol of QX1 (in QIAEX II kit) and 2 vol of water

Add 15 ul of QIAEX II and incubate the mixtures at room temperature for 10 min. Mix every 2 min to keep QIAEX II in suspension

Centrifuge the samples at top speed for 1 min and remove supernatant.

Wash the pellet twice with 500 ul of PE

Air-dry the pellet for 30 min

Add 200 ul of water and respend the pellet. Incubate for 5 min at 50 degree.

Centrifuge for 1 min. Carefully transfer the supernatant into a clean tube.

Centrifuge again for 1 min to remove QIAEX II. Carefully transfer the supernatant into a clean tube.

Precipitate DNA with adding 1/10 vol of 3M Na acetete (20 ul) and 2.5 vol of 95% EtOH (500 ul).

Centrifuge at top speed for 20 min at 4 degree.

Wash the pellet with 500 ul of 70 % EtOH

Air dry the pellet and dissolve it with 20 ul of water

NB: the yield should be about 1-2 ug/750ul crude extract

For BAC end sequencing, you must add a digestion by Eco RV overnight after the RNAse step