Rapid, small-scale isolation of bacteriophage l DNA
Plate Lysate Method
1. Using a sterile pasteur pipette, pick a single, well-isolated plaque into 1 ml of SM containing a drop of chloroform. Store at 4°C for O/N hours to allow the bacteriophage particles to diffuse out of the top agarose.
2. Plate in late afternoon. Mix 100 ml of the bacteriophage suspension (approx. 105 pfu) with 300 ml of indicator bacteria. Incubate for 20 minutes at 37°C. Add 7.5 ml of molten, 0.7% top agarose and spread on the surface of a freshly made, 150 mm agarose plate.
Note: Do not use agar since most batches of agar contain potent inhibitors of restriction endonucleases.
3. Invert the plate and incubate at 37°C O/N until the plaques cover almost the entire surface of the plate.
4. Add 12 ml of SM directly onto the plate and allow the bacteriophages to elute by storing for at least 1-2 hours at room temperature with constant, gentle shaking.
5. Transfer the SM to a 13-ml sarstedt tube, and remove the bacterial debris by centrifugation at 8000g in a fixed angle (J17) rotor for 10 minutes at 4°C.
6. Recover the supernatant, split into 2 sarstedt tubes and add RNase A (final conc. = 10 mg/ml) and DNase I (final conc. = 1 mg/ml). Incubate for 30 minutes at 37°C. (1 ml enzyme/ml SM)
7. Add an equal volume of a solution containing 20% (w/v) polyethylene glycol (PEG 6000-8000) and 2 M NaCl in SM and incubate for 1 hour at 0°C in ice water bath. The PEG/NaCl solution may be prepared in advance and stored at 4°C.
8. Recover the precipitated bacteriophage particles by centrifugation at 8500 rpm in swinging bucket (J13.1) rotor for 20 minutes at 4°C.
9. Pour off supernatant. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away.
10. Add 0.25 ml of SM to each of 2 sarstedt tubes per prep, and resuspend the bacteriopage particles by vortexing.
11. Transfer to microfuge tube and pool individual preps. Microfuge for 2 minute at R.T. to remove debris.
12. Transfer the supernatant to a fresh Eppendorf tube. Add 5 ml of 10% SDS and 5 ml of 0.5 M EDTA (pH 8.0). Incubate at 68°C for 15 minutes.
13. Extract x 2 with phenol/chloroform (1:1), and once with chloroform. Transfer the aqueous phase to a fresh Eppendorf tube between each extraction.
14. To the final aqueous phase add 1/2 volume of NH4Ac and 2.2 vol EtOH. Store at -70°C for 20 minutes. Thaw and centrifuge in an Eppendorf centrifuge for 15 minutes at 4°C.
15. Wash the pellet with 70% ethanol. Dry the pellet and resuspend it in 50 ml of TE (pH 8.0). The DNA may take some time to get into solution.
16. Remove 5 ml of the solution to a fresh Eppendorf tube. Add 2 ml of the appropriate buffer and 1-2 units of the desired restriction enzyme. Incubate for 1.5-2 hours at the appropriate temperature. Store the remainder of the preparation at -20°C (or at 4°C).
17. Add 2 ml RNase. Analyze the DNA fragments in the restriction digest by gel electrophoresis on 0.8% gel.
Notes
1. The addition of DNase-free pancreatic RNase (10 mg/ml) to the restriction enzyme buffer sometimes improves the digestion.
2. The infectivity of DNA prepared in this way and packaged in vitro is identical to that of more highly purified, bacteriophage l DNA.
3. A pop-spin of the eppendorf tube containing the DNA prior to removal of DNA for cutting may aid cutting of DNA (removes contaminants).
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