"
Team:Waterloo/Notebook/Protocols/Miniprep
From 2009.igem.org
1-2-3 Miniprep (Resuspension, Lysis, Neutralization)
Before you start
Prepare solutions 1, 2, and 3. Only solution 2 needs to be made fresh. Solutions 1 and 3 can be made in excess and stored at 4 degrees Celsius.
Solution 1
1. Combine
- 11 mL of 50 mM glucose
- 8.33 mL of 25mM Tris·Cl adjusted to pH 8.0
- 6.67 mL of 10mM EDTA adjusted to pH 8.0
2. Autoclave
3. Add RNase to make the concentration 450 μg/mL
4. Store in 4°C fridge
Solution 2
160 μL MQ water/sample
20 μL 10% SDS/sample
20 μL 2N NaOH/sample
Solution 3
1. Combine
- 60 mL of 5M KAc
- 11.4 mL glacial acetic acid
- 28.5 mL MQ water
2. Store in 4°C fridge
Materials
- Solution 1 (250 μL/sample)
- Solution 2 (250 μL/sample)
- Solution 3 (350 μL/sample)
- 1.5 mL microfuge tubes (3/sample)
- Isopropanol (600 μL/sample)
- 70% ethanol (500 μL/sample)
Instructions
1. Label microfuge tubes.
2. Pour ~1.5mL cell culture in a microfuge tube till it’s almost full.
3. Centrifuge for 30 seconds at 13,000 rpm.
4. Decant the supernatant.
5. Repeat steps 2-4 until all cells have been pelleted.
6. Resuspend cells thoroughly in 250µL of solution 1. Incubate for 2 minutes.
7. Add 250µL of solution 2.
8. Mix gently by inverting 6 to 8 times.
9. Incubate for exactly 5 minutes.
10. Add 350µL of solution 3. Mix gently by inverting 4 to 6 times.
11. Incubate on ice for 5 minutes.
12. Centrifuge for 10 min at 13,000 rpm at 4 degrees Celsius. While waiting, label new sets of tubes.
13. Transfer supernatant to a new 1.5 mL tube. Discard old tubes.
14. Add 600µL of isopropanol. Invert 6-8 times to mix.
15. Spin for 10 min at 13,000 rpm to pellet DNA.
16. Discard supernatant without disturbing pellet.
17. Add 500µL of ice-cold 70% ethanol. Mix gently by inverting 6-8 times to wash.
18. Spin for 10 min at 13,000 rpm to pellet DNA.
19. Carefully decant the supernatant without disturbing the pellet.
20. Leave tubes open in inverted position to dry, or put in speed-vac.
21. Resuspend DNA in MiliQ water for storage in -20 degrees Celsius.
