Using the method of DNA extraction kit

[The genomic DNA extracted from cultured cells]

When using the suspension culture of animal cells:

(1) using the Collection Tube Collection 1 x 105 ~ 1.5 x 107 cell suspension, 5000 RPM centrifuge for 5 minutes, abandon supernatant (cell culture).

(2) to join 150 mu l sterilized distilled water or PBS suspension cells.

(3) to join 500 mu l Solution A and 0.8 mu l RNase A1, intense oscillation for 15 seconds, and then let stand at room temperature for 1 minute.

When using the adherent cells:

(1) that all cultures, to join 650 mu l Solution in A petri dish A, let stand at room temperature for 1 minute.

) 96 orifice plate and so on each hole one hold 650 mu l solution, please fractional processing cell.

(2) with pipetting gun head blown adherent culture cell, take 650 mu l cell suspension was transferred to the Collection Tube.

(3) add 0.8 mu l RNase A1, intense oscillation for 15 seconds, and then let stand at room temperature for 1 minute.

2. Add in 400 mu l Solution B, oscillation mix.

3. Add 1 ml of 4 ℃ precooling Solution C, after thoroughly incorporated, 12000 RPM centrifugal 2 minutes.

4. Left to the upper organic phase, add 1 ml of 4 ℃ precooling Solution C, after thoroughly incorporated, 12000 RPM centrifugal 2 minutes.

5. Left to the upper organic phase, and then transferring aqueous solution (colorless below) to put the Collection Tube Filter on the Cup, 12000 RPM centrifugal for 1 minute.

Note) organic phase (upper) with color, please be sure to do, otherwise it will hinder the DNA binding to DNA preparation of membrane. Interphase precipitation don't have to consider, when the filter will be removed.

6. Put the Filter Cup, add 400 mu in the filtrate l DB Buffer, mixed evenly.

7. The kit http://www.cusabio.com/catalog-10-1.html of the Spin Column to Collection Tube. The above operation 6 mixture transfer to Spin Column, 12000 RPM centrifugal 1 minute, abandon the filtrate.

8. 500 mu l Rinse A join to Spin Column, 12000 RPM centrifugal 30 seconds, abandon the filtrate.

9. 700 mu of Rinse B l join to Spin Column, 12000 RPM centrifugal 30 seconds, abandon the filtrate.

Note) along the Spin around the Column wall to join the Rinse B, this helps to completely flush cleave in the salt on the wall.

10. Repeat steps 9.

11. Will Spin Column placed on the new 1.5 ml centrifuge tube, the Spin of the Column membrane sterilization of the central place in 50 ~ 200 mu l distilled water or Elution Buffer, and let stand at room temperature for 1 minute.

Note) the sterilized distilled water or Elution Buffer when heated to 65 ℃ is used to improve the efficiency of Elution.

12. 12, 000 RPM centrifugal 1 minute elution DNA.