Small-scale Dialyzed Phage Genomic DNA Prep from Mycobacteriophage Lysate – Agarose Plates

Gregory Broussard

Revision: 07/29/11

Reagents:

Saturated ammonium sulfate: Bring 80 g ammonium sulfate to 100 ml in dH₂O, and stir for > 2 hrs on stir plate. Allow the undissolved crystals to settle. Remove solution, leaving behind undissolved crystals, and place in 50 ml conical tubes.

95% EtOH

70% EtOH

Buffer-equlibrated phenol

Phenol:chloroform:isoamyl alcohol (25:24:1)

Chloroform

RNAse A (10mg/mL)

DNase I (2,000 U/ml)

TE

7H9+agarose plates

MBTagarose

Methods:

1. Prepare plates of 7H9 (4.7g), agarose (15g), 10 ml 0.1 M CaCl₂, 2-3 drops antibubble and 12.5 ml 40% glycerol in 900ml H₂0; add ADC, CHX, and CB after autoclaving.  (The use of agarose instead of agar remedies a problem of co-purification of a substance from the agar that inhibits some restriction enzymes and possibly other types of enzymes).  Also, prepare MBTagarose by adding agarose in the place of agar in the MBTA recipe.
2. Plate dilutions of phage on normal 7H10 plates to determine the dilution that gives you “webbed” plates.
3. Plate for “webbed” plates of your phage: use 7H9+agarose plates and MBTagarose. (4 plates is usually appropriate)
4. Prepare a lysate and filter (0.22 mm).
5. Add an equal volume of saturated ammonium sulfate, invert tube to mix gently, and incubate on ice 2 hrs.
6. Pellet at 3500 x g for 10 min. at 4°C in a 50 ml conical tube. Mark the tube where the pellet is expected. Pellet this twice, but do not remove the supernatant after the first spin!!! The first time, the phage are in “chunks” of glassy pellet that you can see when inverting the tube gently. The second time, the pellet is more visible. Immediately remove the supernatant the second you take it out of the centrifuge. (If no pellet is seen, try centrifuging for 30 min.)
7. Resuspend phage in 500 ml Phage buffer.
8. During the above centrifugations, soak ~4” of dialysis tubing (15,000 MWCO) in 1L diH₂O (for ~10 min.)
9. Dialyze your 500 ml phage sample at 4°C with stirring, twice in phage buffer: once overnight and once for two hours (the order does not matter).
10. Transfer sample to a 1.5 ml tube (unless volume is >700ml, then use 2 tubes).
11. Check the volume and add the appropriate amount of 10X DNase I reaction buffer (final conc. 1X).
12. Add 1ml of DNase I (2,000 U/ml; total of 2U) and X ml RNase A (10mg/ml; final conc. 100mg/ml) and incubate at 37°C for 1 hr. (DNase I will remove trace amounts of M. smeg DNA, the RNase A removes precipitated M. smeg ribosomes)
13. Add an equal volume of buffer equilibrated phenol, mix until milky white, centrifuge at 13K rpm at RT for 5 min.
14. Save aqueous phase, repeat phenol extraction until white interface is gone (~3-5 times total).  Also, back-extract with 600 ml TE.
15. Add equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix until milky white, centrifuge as above.
16. Save aqueous phase and add equal volume of chloroform, mix and centrifuge as above.
17. Save aqueous phase, add 1/10 vol. 3M sodium acetate and 3 vol. 95% ethanol.  Mix gently and well by inverting tube repeatedly until DNA precipitates and forms a nice “cotton ball”. (Do Not Vortex)
18. Freeze on dry ice.  Thaw on top of ice.  Centrifuge at 14K rpm at 4 °C for 30 min.
19. Remove ethanol leaving pelleted DNA in tube.  Add 1 ml 70% ethanol and mix gently by inverting tube 6-8 times (pellet should float around in ethanol).  Centrifuge at 13K rpm at RT for 1 min.
20. Remove ethanol, pipette out any droplets, and air dry (~20 min.).  Don’t let the DNA over dry or it will be hard to dissolve later, but do make sure all the ethanol has evaporated.
21. Dissolve DNA in TE at 42°C for ~10 min. or until dissolved.  Pipette gently to resuspend DNA.  (Start with 50 ml TE, then add more if needed.  It may be a good idea to add no more than 100 ml TE and leave it at RT overnight, then add more TE if needed.) Store at 4 °C.
22. Measure the concentration of DNA on the nanodrop, measure undiluted DNA twice and 1:10 dilution once, make sure all readings are consistent.

Notes:

1. This procedure will yield ~50-150 µg DNA
2. Titers of dialyzed phage equal 10¹⁰ to 10¹² pfu/ml
3. Transformation of M. smeg with 100 ng BPs DNA yielded 10⁴ pfu/µg
4. PCR on BPs DNA gave product with BPs specific primers and no product with M. smeg specific primers
5. BPs DNA gave correct band patterns when digested with BamHI, ClaI, EcoRI, HaeIII, KasI, SacI, NotI
6. Dialyzed phage treated with RNase A has NOT been used at this time for EM work