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Team:Waterloo/Notebook/Protocols/Miniprep
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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.
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 (100 μL/sample)
- Solution 2 (200 μL/sample)
- Solution 3 (150 μL/sample)
- 1.5 mL microfuge tube (3/sample)
- Chloroform (150 μL/sample)
- 95% ethanol (2 mL/sample)
- 70% ethanol (150 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 about 3 times.
6. Resuspend well in 100µL of solution 1.
7. Add 200µL of solution 2.
8. Mix gently by inverting 6 to 8 times.
9. Add 150µL of solution 3.
10. Mix gently by inverting 4 to 6 times.
11. Centrifuge for 5 min at 13,000 rpm. While waiting, label new sets of tubes.
12. Transfer supernatant to a new 1.5 mL tube. Discard old tubes.
13. Add 150µL chloroform.
14. Vortex until well mixed.
15. Spin for 3 min at 13,000 rpm. Label new sets of tubes.
16. Transfer top aqueous layer to a new tube. Discard old tubes.
17. Add 2× (~800µL) of ice-cold 95% ethanol.
18. Vortex.
19. Centrifuge at 13,000 rpm for 20 minutes.
20. Do not disrupt pellet. Carefully draw off the supernatant with a pipette and discard to liquid waste.
21. Rinse with 150 µL ice-cold 70% ethanol.
22. Dry tubes open in the 37ºC for 10 minutes.
23. Resuspend DNA in 50 µL MQ water.
25. Check concentration with Nanodrop.
