Carefully remove the centrifuge tube from the rotor


It is necessary to use this unique rotor. 341 650g (opt […]

It is necessary to use this unique rotor. 341 650g (optima l-90k ultracentrifuge), centrifuged at 18C for at least 8h. It can be centrifuged overnight.
1. Carefully remove the centrifuge tube from the rotor, taking care not to damage the gradient. A 23g needle was used to perforate the top of the centrifuge tube. With UVB, carefully remove the glowing DNA strip with an 18G needle with an upward slope. There are usually two bands. Select the strip at the bottom.

The top band is degraded DNA, while the bottom band is complete circular bacdna. The volume of the strip removed is about 200 UL. Don't take more than 200 UL, or you'll get - part of the top degradation band. Transfer the DNA to a 15 ml Falcon tube and replenish the total volume to 2 ml with 1 xTe buffer.
2. Add equal volume of NaCl saturated butanol into DNA solution, and mix gently. Let the mixture stand for 30 s to separate. Remove and discard the upper layer. Extract the solution 4-5 times until there is no pink color.

3. Transfer DNA solution into a 30 ml round bottom centrifuge tube, add 1 ml dh2o into the solution, and then add 2.5 ~ 3.0 volume of 100% ethanol. They were mixed and incubated at - 20'c for 30 min. 16417g, centrifuged at 4'C for 30min to precipitate DNA (j-25i Beckman Avanti centrifuge, ja-25.50 rotor).
4. Pour out the ethanol and re suspend the DNA in 0.5 ml of 0.3 mol / L sodium acetate. Transfer the solution to a 1.5 ml micro centrifuge tube and add 1 ml of 100% ethanol. The solution was centrifuged at 20 617 g at 4'C for 30 min.
Because BAC DNA is sensitive to shear force, a pipette with a wide hole suction head should be used to transfer DNA.