$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
1. Purification of Phage
NOTE: Sucrose purification is largely dependent on the size of the phage. Considerations for the mass of the phage to be isolated will have to be made, and adjustments to gradient conditions will be performed. Final viral titers of over 1013 phage/mL are easily achievable with this method.
- Prepare 12 mL of 5% and 45% w/v sucrose in 1x TM buffer.
- Prepare 4x 5 45% sucrose gradients by pipetting 2.5 mL of 45% sucrose into 4 ultracentrifuge tubes. Top with ~ 2.6 mL of 5% sucrose, stopping when the liquid reaches 2 mm from the rim of the tube.
NOTE: Place the pipette tip into contact with surface of the sucrose solution and very slowly dispense liquid. The lighter sucrose solution will float on top of the heavier one, forming a distinct boundary. A properly layered solution will have ~1 mm interface if held against background light. - Mix the gradients by tilt-tube rotation using a gradient forming instrument for 43 s at 86° at 23 rpm. If not immediately needed, cover with paraffin film and store at 4 °C.
- Remove 500 µL from the top of each prepared sucrose gradient with a 1 mL pipettor and balance to within ± 0.002 g.
- Pool phage suspensions from all tubes. Using a 1 mL pipette, add 500 µL of the suspension by bringing the tip into contact with the liquid surface and very slowly dispensing the volume onto the top of the sucrose gradient, being careful not to mix through rapid pipetting. Centrifuge at 70,000 x g for 20 min at 4 °C.
NOTE: Balance the prepared gradients to within ± 0.002 g. Smaller phages may take up to 1 h. - Remove the phage bands using a sterile syringe and blunt cannula.
NOTE: When viewed from the side, the phage band will be thick and milky compared to the surroundings, approximately 5 mm in height, and situated half way down the centrifuge tube.- Submerge a blunt cannula into the solution until the tip is centered on the very upper edge of the phage band. Remove the band by drawing on the syringe plunger until the majority of the phage band has been removed.
- Add the sucrose volume with the suspended phage to 3-4 ultracentrifuge tubes. Add no more than 1.5 mL/tube. Fill to ~3-4 mm from the top of the tube with cold 1x TM. Cover with paraffin film and invert to mix. Place back in an ultracentrifuge and spin at 145,000 x g for 1 h at 4 °C to pellet. Smaller phages may take up to 2 h.
- Quickly pour off the supernatant and drain upside down on disposable wipes. Wipe the inside of the tube with a sterile cotton swab to remove excess supernatant.
- Divide 200-400 µL cold 1x TM across all pellets and resuspend overnight at 4 °C; no shaking is required.
NOTE: If the pellet is not clean, e.g., stringy bits of membrane, DNA, etc., pool and perform additional centrifugation in a microcentrifuge at 17,000 x g.