DNA Preparation, Plasmid DNA - Qiagen Method

Jeff Lawrence

(This protocol uses solutions provided with the Qiagen Kit)

1. Grow 5 ml culture overnight in appropriate antibiotic. Inoculate 100-250 ml of fresh media with 3-5 ml culture and grow to Klett 150 : end log growth.

2. Centrifuge cells at 8 krpm for 10 min in GSA rotor. Discard supernatant and resuspend pellet in 30 ml TE. Centrifuge at 10 krpm for 8 min in SS34 rotor.

3. Discard supernatant and resuspend pellet in 4 ml of buffer P1. Add 4 ml af buffer P2 and incubate at room temperature for 5 min.

4. Add 4 ml of buffer P3, mix gently, and centrifuge at 12 krpm for 30 min at 4°. Decant supernatant and save.

5. Equilibrate Qiagen-tip 100 with 3 mL buffer QBT and allow to empty.

6. Apply the supernatant from Step 4 to the column and allow to empty. Apply 10 ml buffer QC and allow to empty.

7. Elute the DNA with 5 mL buffer QF and collect in a 15 mL Corex tube.

8. Add carrier; mix well. Add 5 mL Isopropanol at room temperature; mix well. Centrifuge at 12 krpm for 60 min at 4°. Discard supernatant.

9. Add 10 mL 70% Ethanol. Rinse pellet well. Microfuge for 2 min; discard the supernatant.

11. Dry pellet for 4-10 min in vacuum desiccator. Resuspend pellet in 1 mL TE.

Last Update: Thursday, 19-Jun-2014 11:39:20 PDT
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Eric Kofoid eckofoid at ucdavis.edu