Team:Paris Saclay/mini maxi


Plasmidic DNA Mini and Maxi preparation

1. Centrifuge during 1 minute the cell culture in tubes of 1.5 or 2.0 mL at maximal speed in a benchtop centrifuge

2. Add 100µL of solution n°1 (50 mM glucose, 10 mM EDTA and 25 mM Tris pH 8.0) and resuspend the cells

3. Add 200µL of solution 2, the lysis solution(0.2 mM NaOH et 1% SDS), mix the final solution until it is translucent

4. Add 150µL of solution 3 (potassium acetate 3.3M)to precipitate cell debris, genomic DNA and proteins, and incubate in ice during 10min

5. Centrifuge at maximal speed during 10 min in a benchtop centrifuge

6. Take off the liquid supernatant and transfer it in a new 1.5 ml tube

7. Add 500µL of phenol/chloroform/isoamyl alcohol 25/24/1 and mix vigorously

8. Centrifuge at maximal speed during 8 min, take off the aqueous phase and transfer it in a new 1.5 ml tube

9. Precipitate the plasmidic DNA with ethanol (see protocol)

For Maxi Preparation, the protocol was scaled up for cultures of 50 or 100 ml.