Team:USTC CHINA/Notebook/Protocols/Plasmid mini-prep


Plasmid mini-prep

Performed with Shanghai Sangon Plasmid miniprep kit.
1. Preparation
1) Make sure the RNase A has been added into Buffer P1;
2) Make sure the ethanol has been added into Wash Solution;
3) Make sure there is no deposit in Buffer P2 and P3;
2. Collect the deposit of bacteria from 1.5ml~5ml bacteria liquid (centrifuge at 8,000×g for 2 min).
3. Add 250μl Buffer P1 into the deposit and suspend it thoroughly.
4. Add 250μl Buffer P2, mix gently and keep standing in room temperature for 2~4min.
5. Add 350μl Buffer P3 and mix gently for 5~10 times.
6. Precipitate the bacteria fragments (centrifuge at 12,000×g for 5~10 min) and extract the supernatant (centrifuge at 8,000×g for 30s).
7. Add 500 μl Buffer DW1, centrifuge at 9,000×g for 30s
8. Add 500 μl Wash Solution, centrifuge at 9,000×g for 30s. Repeat this process for one more time. Centrifuge the empty bottle at 9,000×g for 60s.
9. Add 50~100μl Elution Buffer and collect the plasmid.