SOP: ABC-LP-017
Prepared by: Marleen Cook
Version: v.1
Date: 03MAR2013
Title: DNA Isolation
PURPOSE: The purpose of this SOP is to guide students on proper DNA Isolation
technique.
SAFETY CONSIDERATIONS: Proper PPE should always be worn this includes lab coats,
gloves, safety glasses, closed toe shoes and no mouth pipetting. All materials should be
treated as a biohazard and should be properly disposed of by proper aseptic techniques.
MATERIALS:
Pipettes and pipette tips
Laboratory spatula
MicroBead Tube
Microbead Solution
Solution MD1
Solution MD2
Solution MD3
Solution MD4
Solution MD5
MOBIO Vortex Adapter Tube Holder
Centrifuge
4⁰C Refrigerator
MATRIX/SPECIMEN: Fungal Colony provided by K-State.
SPECIMEN ID, HANDLING AND STORAGE: Fungal colony must be present in nutrient
petri dish and stored according to fungal colony SOP.
SAMPLE PREP: N/A
PROCEDURE:
1. Scrape fungal colony and place in MicroBead Tube.
2. Check Solution MD1. If Solution MD 1 is precipitated, heat the solution at 60  C
until the precipitate has dissolved.
3. Add 50 l of Solution MD1 to the MicroBead Tube.
4. Add 300 l of MicroBead Solution to Microbead Tube.
5. Secure MicroBead Tubes horizontally using the MOBIO Vortex Adapter tube
holder for the vortex (MO BIO Catalog# 13000-V1) or secure tubes horizontally
on a flat-bed vortex pad with tape.
6. Set vortex at maximum speed for 10 minutes.
7. The 2 ml MicroBead Tubes must rotate freely in the centrifuge without rubbing.
Centrifuge the tubes at 10,000 x g for 30 seconds at room temperature
CAUTION:Be sure not to exceed 10,000 x g or tubes may break.
8. Transfer the supernatant to a clean 2 ml Collection Tube. (Expect 300 to 350 l
of supernatant)
9. Add 100 l of Solution MD2, to the supernatant.
10. Vortex for 5 seconds.
11. Incubate at 4 C for 5 minutes.
12. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
13. Avoiding the pellet, transfer the entire volume of supernatant to a clean 2 ml
Collection Tube provided (Expect approximately 450 l in volume).
14. Mix Solution MD3 by shaking. Add 900 l of Solution MD3 to the supernatant
and vortex for 5 seconds.
15. Load approximately 700 l into the Spin Filter and centrifuge at 10,000 x g for 30
seconds at room temperature.
16. Discard the flow through and add the remaining supernatant to the Spin Filter.
17. Centrifuge at 10,000 x g for 30 seconds at room temperature.
NOTE: A total of 2 to 3 loads for each sample processed are required.
18. Discard all flow through liquid.
19. Add 300 l of Solution MD4 and centrifuge at room temperature for 30 seconds
at 10,000 x g.
20. Discard the flow through.
21. Centrifuge at room temperature for 1 minute at 10,000 x g.
22. Being careful not to splash liquid on the spin filter basket, place Spin Filter in a
new 2 ml Collection Tube.
23. Add 50 l of Solution MD5 to the center of the white filter membrane.
24. Centrifuge at room temperature for 30 seconds at 10,000 x g.
25. Discard Spin Filter.
26. DNA in the tube is now ready for any downstream application.
ACCEPTANCE FOR RESULTS: Proper technique will be evident in future experiment.
ANALYSIS AND INTERPRETATION OF DATA: N/A
REFERENCES: UltraClean DNA Isolation Kit.