DNA Purification

Complete the following steps to perform DNA purification, the final step to extract DNA before library preparation.

Always cap and seal tubes before centrifuge steps.

• Genomic Lysis Buffer
• Urine DNA Prep Buffer
• Urine DNA Wash Buffer
• MBG (Molecular Biology Grade) Water

Labeled Tube Sets from UCB Treatment Preparation:

• Zymo-Spin III-Filter
• Zymo-Spin IC-S Column
• 2.0 ml collection tubes
• 1.5 ml microcentrifuge tube
• 2.0 ml microcentrifuge tube
1. Place Zymo-Spin III-Filter into a new labeled 2.0 ml microcentrifuge tube.
2. For each sample, transfer 400 µl homogenate lysate from step 6 of Sample Homogenization and Cell Lysis onto the Zymo-Spin III-Filter.

Avoid disturbing the bead volume.

3. Centrifuge at 10,000 × g for 1 minute.
4. Discard the Zymo-Spin III-Filter. Keep the flow-through.
5. Add 1.2 ml Genomic Lysis Buffer to each sample flow-through.
6. Vortex briefly to mix, and then centrifuge briefly.
7. Place Zymo-Spin IC-S Column into a new labeled 2.0 ml collection tube.
8. Transfer 800 µl of mixture to the Zymo-Spin IC-S Column.
9. Centrifuge at 10,000 × g for 1 minute.
10. Transfer Zymo-Spin IC-S Column to a new labeled 2.0 ml collection tube.
11. Discard the collection tube containing the flow-through.
12. Transfer the remaining sample filtrate volume from step 5 to the Zymo-Spin IC-S Column.
13. Centrifuge at 10,000 × g for 1 minute.
14. Transfer the Zymo-Spin IC-S Column to a new labeled 2.0 ml collection tube.
15. Discard the collection tube containing the flow-through.
16. Add 200 µl Urine DNA Prep Buffer to the Zymo-Spin IC-S Column.
17. Centrifuge at 10,000 × g for 1 minute.
18. Transfer Zymo-Spin IC-S Column to a new labeled 2.0 ml collection tube.
19. Discard the collection tube containing the flow-through.
20. Add 700 µl Urine DNA Wash Buffer to the Zymo-Spin IC-S Column.
21. Centrifuge at 10,000 × g for 1 minute.
22. Transfer Zymo-Spin IC-S Column to a new labeled 2.0 ml collection tube.
23. Discard the collection tube containing the flow-through.
24. Add 200 µl Urine DNA Wash Buffer to the Zymo-Spin IC-S Column.
25. Centrifuge at 10,000 × g for 1 minute.
26. Transfer Zymo-Spin IC-S Column to labeled 1.5 ml microcentrifuge tube.
27. Discard the collection tube containing the flow-through.
28. Add 70 µl MBG water to Zymo-Spin IC-S Column.
29. Incubate for 5 minutes at room temperature.
30. Centrifuge 1.5 ml microcentrifuge tube at 10,000 × g for 1 minute.
31. Reapply the elute (70 µl) to the Zymo-Spin IC-S Column.
32. Incubate for 5 minutes at room temperature.
33. Centrifuge 1.5 ml microcentrifuge tube at 10,000 × g for 1 minute.

The final eluted volume contains purified sample DNA.