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.
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