Team:Waterloo/Notebook/Protocols/Miniprep

 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.