DNA from P22 heads.

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From Youderian, Sugiano, Brewer, Higgins and Elliot. 1988.
Packaging specific segments of the Salmonella chromosome
with locked-in mud-P22 prophages. Genetics. 118: 581-92.

Modified by Kim Bunny, July 1999

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1. Grow O/N culture.

2. Dilute culture 1/100 into 30 mLs LB, shake at 37¡C till the cell density reaches 5x10⁸ cells per mL (3-4 hours).

3. Add fresh Mitomycin C to a concentration of 2 µg/mL.

4. Shake at 37¡C until lysis is visible (generally O/N).

5. Spin out cell debris (6000 rpm, 15 mins), transfer supernatant to a new tube.

6. Pellet phage particles (17000 rpm, 120 mins).

7. Resuspend phage in 1/10th volume of PBS (3 mLs).

8. To 3 mLs of PBS add 2 µl DEPC, 10 µl 10% SDS, 50 µl 2M Tris-HCl, 0.5 M EDTA (pH 8.0). Leave at 65¡C for 15 mins.

9. Add 50 µl KCl, incubate on ice for 1 hour.

10. Pellet protein/SDS complex by spinning 15 mins in a microfuge (13000g).

11. Precipitate DNA by adding 2 volumes of ethanol and incubating at -70¡C for 20 mins or overnight at 4¡C.

12. Spin 15 mins in microfuge. Dry and resuspend pellet in 200 µl 10 mM Tris-HCl (pH 8.0).

13.Treat the isolated DNA with proteinase K to remove any associated phage head proteins (add 3 µls 20 mg/mL proteinase K and leave at 37¡C for 30 mins).

14. Extract once with phenol/chloroform/isoamyl alcohol (25:24:1).

15. Ethanol precipitate (add 1/10th volume 3 M Na acetate and two new volumes of ethanol, incubate as above).

16. Spin, dry and resuspend the pellet in 300 µl H₂O.

17. For sequencing 1 µg of DNA in 6 µl is required.

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Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu