DNA Preparation - Mud-P22 DNA
DNA Preparation - Mud-P22 DNA

Jeff Lawrence

1. Grow an overnight culture of a strain with a MudP or MudQ hybrid Mud-P22 insertion.

2. Transfer 1 mL of culture to 30 mL LB. Grow to a density of 5x108 cells/mL at 37 C.

3. Add Mitomycin C to 2 µg/mL. Allow culture to incubate at 37 C an additional 2 hr.

4. Add 500 µl Chloroform. Incubate at 37 C for 10 min with shaking.

5. Centrifuge at 8000 rpm for 15 min in an SS34 rotor. Transfer lysate to a fresh tube.

6. Centrifuge at 8000 rpm for 10 min. Transfer clear lysate free of any debris to a fresh tube.

7. Centrifuge at 17000 rpm for 2 hr. Discard supernatant.

8. Resuspend bacteriophage particles in 500 µl Buffer Saline.

9. Add 500 µl Chloroform. Mix well. Centrifuge for 2 min. Transfer supernatant to a fresh tube.

10. Add 5 µl of 100 mg/mL DNase and 5 µL of 1 mg/mL RNase. Incubate at 37 C for 30 min.

11. Add 500 µl Buffered Phenol. Mix well by vortexing. Centrifuge for 10 min at 4 C. Transfer supernatant to a new tube.

12. Add 500 µl 24:1 Chloroform:Isoamyl alcohol. Mix well by shaking. Centrifuge for 3 min at 4 C. Transfer supernatant to a fresh tube.

13. Add 1 mL 95% Ethanol and mix. Incubate at -20 C for 2 hr. Centrifuge for 15 min at 4 C. Discard supernatant.

14. Add 1 mL 70% Ethanol. Mix well to dislodge the pellet. Centrifuge for 3 min at 4 C.

15. Discard supernatant. Air dry. resuspend pellet in 50 µl TE.



Last Update: Thursday, 19-Jun-2014 11:40:48 PDT
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Eric Kofoid eckofoid at ucdavis.edu