Phage DNA Extraction

Bacteriophage DNA Miniprep

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.

Phage genomic DNA extraction – modified Promega Wizard method

In this protocol you extract the genomic DNA from the phages in a lysate. The lysates are “dirty” in that they contain spent media components, cell wall debris, flagella, nucleic acids, bacterial proteins and unassembled phage proteins in addition to the phage themselves. There are many methods for purifying phage DNA from the rest of the lysate, and in this protocol we will use a modified method with commercial DNA extraction kit, the Promega Wizard DNA Clean-up kit (Promega catalog #A7280).

As a rule of thumb, 1 ml of phage lysate with a titer of 1 x 10^10 contains about 0.5 μg of phage DNA, assuming the phage has a 50 kb genome. Typically 10 ml of a high-titer phage lysate is used for DNA extraction. More or less lysate (up to 20 ml) may be used depending on the phage stock titer and the expected genome size of the phage. For low-titer phage stocks, up to 20 ml of lysate may be used. With high titer stocks of large-genome phages, use 10 ml of lysate or less or else the column will be overloaded and clog. The columns have a maximum capacity of about 30 μg of DNA.

Reagents required
Promega Wizard DNA cleanup kit (cat# A7280)
Phage lysate (10-20 ml, >109 PFU/ml)
Nuclease solution (20 mg/ml DNase I, 20 mg/ml RNase A)

Precipitant solution (30% w/v PEG-8000, 3 M NaCl)
Preparation (200 ml): in a clean bottle add 110 ml ddH2O and 35 g NaCl, dissolve. Add 60 g PEG-8000, cap bottle and shake. Incubate bottle in a 50 – 60 oC water bath for about 2 h, shaking occasionally. Remove and let cool to RT, shaking occasionally. Solution should be clear or slightly turbid. Add ddH2O to 200 ml, store at RT.

Resuspension buffer (5 mM MgSO4 in water)
80% (v/v) isopropanol
Sterile molecular biology-grade water

Materials required
50 ml centrifuge tubes
Sterile 1.5 and 2 ml microcentrifuge tubes
3 cc syringes
Heat block, 80 oC

1. Innoculate 2 ml LB with E coli K12 ER1793, grow to log phase.

2. Make LB agar, pour LB top agar containing 0.1 ml of Ecoli K12 ER1793.

3. Grow o/n at 37 oC.

4. Dissolve T4 phage in 10 ul water, add to the center of LB/agar/top agar.

5. Incubate at 37 oC until the bacteria is completely lused.

6. cut out one small piece of top agar, add to 5 ml fresh cultured E coli K12 ER1793.

7. Grow o/n at 37 oC.

8. Spin down cell debris from lysates, 6000 rpm for 3 min. Add 1 ul of Nuclease solution  and incubate for 30 min at 37 oC.

9. Add precipitant solution to the lysate at a rate of 1:2 precipitant:lysate (10% PEG-8000, 1 M NaCl final). Mix gently by inversion. Incubate on ice for at least 1 hr; 1 hour is enough for T4 phage

Precipitation works best when incubated at 4 oC overnight. Most phages are stable in this state for up to several days.

Alternative method: phages may be pelleted from the lysate by centrifugation in a normal high-speed centrifuge (e.g., about 7,000 x g overnight) or in an ultracentrifuge (e.g., about 1 hour at 50,000 x g) without PEG+NaCl.

dsDNA phages have weights of about 400-1000 S, and pelleting time (in hours) can be calculated from the rotor’s k-factor by the formula:

time = k/S.
Type 35, K=225

10. Centrifuge the precipitated phage lysate at 10,000 x g, 4 oC, 10 min.

or Type 35: 25000 rpm is 50,000 x g. Spin for 20 hour without incubating o/n.

11. Carefully pour off the supernatant and retain the pellet. The pellet may be transparent or opaque, and may be spread up the wall of the tube.

12. Resuspend the pellet in 2X 500 μl of resuspension buffer (5 mM MgSO₄) by pipetting gently up and down; be sure to rinse down the sides of the tube to obtain all of the pellet. For extraction of DNA, water can be used to resuspend phage pellet.

13. Transfer the resuspended phage to a labeled 1.5 ml microcentrifuge tube.

Optional step: some bacteria, such as Staphylococcus aureus, produce heat-stable nucleases that are resistant to denaturation. The nuclease will be present in the phage precipitate and degrade the phage DNA once it is released from the phage capsid. In these cases, the nuclease can be degraded by addition of proteinase K. To each 500 μl aliquot of resuspended phage, add 10 μl of 0.5 M EDTA pH 8 and proteinase K to a final concentration of 100 μg/ml. Incubate at 50 oC for 30 min. Allow the tube to cool to RT and continue to the next step.

14. Use genomic DNA spin column kit to purify phage DNA from 100 ul suspension. Elute DNA using 100 ul elution buffer

Lane 1 & 2 are T4 phage genomic DNA