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Star Republic: Guide for Biologists

Mini plasmid prep (Qiagen)

Qiagen purified DNA usually is good enough for transfection and micro-injection.

1. Grow 1-5 ml of bacteria culture for one mini prep.

3. Chill P3 on ice.

4. Centrifuge to pellet cells.

5. Discard supernatant, and resuspend the bacterial pellet in 0.3 ml P1/RNAse A by vortexing.

6. Add 0.3 ml P2, mix by inverting 4-6 times, and incubate at room temperature for 5 minutes.

7. Add 0.3 ml of P3, mix by inverting 4-6 times. Incubate on ice for 5 minutes.

8. Centrifuge at maximum speed in a microfuge for 10 minutes. Remove supernatant to a fresh tube promptly.

10. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow.

11. Add the supernatant containing DNA to the QIAGEN-tip and allow it to empty by gravity flow.

12. Wash the QIAGEN-tip with 1 ml Buffer QC.

13. Repeat step 12 three times.

14. Connect the QIAGEN-tip with a 1.5 ml microcentrifuge tube. Elute DNA with 0.8 ml Buffer QF.

15. Precipitate DNA by adding 0.7 volumes of room temperature isopropanol. Mix and centrifuge at maximum speed in a microfuge for 30 minutes. Carefully discard supernatant.

16. Wash DNA pellet with 1 ml 70% ethanol. Carefully decant the supernatant without disturbing the pellet.

17. Allow the pellet to air dry. Resuspend the DNA in 20-50 ml of TE or ddH2O.