1. LB
2. Agar plate with transformed E. coli colonies grown on it or glycerol stock
3. Test tube or 50ml disposable centrifuge tube
4. Incubater shaker
5. Qiagen Plasmid Mini Kit
6. Pippetters and tips
7. Bech-top centrifuge

1. Grow 10 ml culture from a single colony of transformed E. coli (alternitvely 50µl glycerol stock but not recommended) by incubating at 37^oC and shaking at 300rpm overnight.
2. Transfer 1.5 ml overnight culture into an eppendorf tube. Spin at 10,000rpm for 1min. Discard the supernatant. If the pellet is too small, add another 1.5 ml culture and spin again until there is enough pellet.
3. Add 250 µl Buffer P1 solution (in 4^oC refrigerate) containing RNase A resuspend evenly by pippetting or vortexing vigorously.
4. Add 250 µl Buffer P2 solution (room temperature) and mix by inverting the tube 4-8 times. no vortexing! Lyse cells at room temperature for 5 min.
5. Add 350 µl Buffer N3 and invert the tube immediately 4-8 times to neutralise the lysis reaction.
6. Spin at 13,000 rpm for 10 min.
7. Apply the supernatant to the QIAprep spin column by decanting.
8. Spin for 30–60 s. Discard the flow-through.
9. Wash the column by adding 600 µl Buffer PE and spin for 30 second at 13,000 rpm. Discard the flow-through.
10. Wash again with 200 µl Buffer PE and spin for 1 min at 13,000 rpm. Discard the flow-through and put the column back in a different rotation angle so that there will be no solution retentate in the corner of the column. Spin for an additional 1 min to remove residual wash buffer.
11. Place the column in a clean sterile Eppendorf tube.
12. To elute DNA, add 50 µl Buffer EB to the center of the column, let stand for 1-5 min, and centrifuge for 1 min at 13,000 rpm.
13. Store DNA solution at 4^oC

Reading:

QIAprep Spin Miniprep Kit manual