Plasmid DNA Miniprep Kit Protocol
Before you start:
Add the provided RNasa A solution to Buffer MX1, mix and store at 4 C.
Add 98-100% ethanol to Buffer WS before use (see bottle label for volume).
I.
Using a Centrifugee:
1. Grow 1-5 mL plasmid-containing bacterial cells in LB medium w/
appropriate antibiotic(s) (amp in this case) overnight (12-16 hours) on a
shaker.
2. Pellet the cells by centrifuging 1 mL for 5 minutes at 8000 rpm
3. Resuspension: Add 250µL MX1 Buffer to the cell pellet.
~Vortex so there are no cell clumps
4. Lysis: Add 250µL MX2 Buffer and gently invert the tubes 4-7 times.
~Do NOT vortex. Let rest on bench about 5 min.
5. Neutralize: Add 350 µL MX3 Buffer and a white precipitate should form.
Do NOT vortex.
6. Centrifuge for 10 min. at 13000 rpm. Meanwhile, place the columns onto
a collection tube.
7. Transfer the supernatant carefully to the column.
8. Centrifuge 30 seconds at 5000 rpm. Discard the flow-through.
9. Wash: Wash the column once with 500µL WF Buffer by centrifuging 30
seconds at 5000 rpm. Discard the flow-through.
10. Wash: Wash the column once with 750µL WS Buffer by centrifuging for
30 seconds at 5000 rpm. Discard the flow-through.
11. Centrifuge the column at 12000 rpm for 1 minute to remove residual
ethanol. *Important
12. Place the column onto new 1.5mL centrifuge tube. Add 50 µL of Elution
Buffer onto the center of the membrane.
13. Stand the column for 2 minutes at room temp. and centrifuge for 30
seconds at 12000 rpm and elute DNA.
14. Store plasmid DNA at 4 C or -20  C.