DNA Clean Protocol For the Genomic DNA Clean & Concentrator®-5 kit

Buffer Preparation

• Before starting: Add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA Wash Buffer concentrate.
• Add 192 ml 100% ethanol (208 ml 95% ethanol) to the 48 ml DNA Wash Buffer concentrate.

Sample Processing

All centrifugation steps should be performed between 3,500 – 5,000 x g

1. Warm DNA elution buffer (10mM tris HCL) to 70 Celsius degrees.
2. Transfer 20-100 ul of DNA into a 1.5 ml tube.
3. Add double the DNA volume (40-200 ul) of DNA binding buffer to each tube.
4. Vortex briefly to mix thoroughly.
5. Transfer the mixture (60-300 ul) to a Zymo spin column.
6. Centrifuge for 30 sec at 3,500 – 5,000 x g.
7. Discard the flow-through.
8. Add 40-200 ul of DNA wash buffer to the column.
9. Centrifuge for 30 sec at 3,500 – 5,000 x g.
10. Add 40-200 ul of DNA wash buffer to the column.
11. Centrifuge for 30 sec at 3,500 – 5,000 x g.
12. Discard the flow-through.
13. Transfer the column to a 1.5 ml microcentrifuge tube.
14. Add 20-100 ul of warmed DNA elution buffer (10mM tris HCL) directly intro in the column matrix.
15. Incubate at Room temp x 1 min.
16. Centrifuge for 30 sec at 3,500 – 5,000 x g to elute the DNA.
17. Ultrapure DNA is now ready for use.

QC

Qubit: Broad Range DNA

• Follow Qubit protocol
• Read samples twice and average readings
• Make sure to record standards

DNA Gel

• Follow Gel Protocol
• Suggestions are to:
  • Make a 1% gel and run for 1hr at 100V
  • Use 3ul 1kb plus DNA ladder
  • Use gel green