From 2007.igem.org

Growing colonies in broth:

• 1. Prepare 5mL LB + Amp broth in a sterile Falcon tube.
• 2. Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon.
• 3. Incubate at 37C incubator for 14-16 hours.

Miniprep Plasmid Isolation (with Qiagen kit):

• 1. Centrifuge the falcons at 4000rpm for 4-10 minutes.
• 2. Resuspend pelleted bacterial cells in 250ML Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.
• 3. Add 250ML(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).
• 4. Add 350ML Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.
• 5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.
• 6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.
• 7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.
• 8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.
• 9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32ML (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C center of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.

Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.

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Digestion