Gill:CsCl step gradient for phage K

Cesium Step-Gradient Purification

Phage growth (optimized for Bacteriophage K)

1. Grow 3ml overnight culture of bacterial host

2. Inoculate 25-250ml culture (depending on how much is needed) at 1:100

3. Grow at 30C to OD = 0.5

4. Infect at MOI= 2-5, incubate until culture clears

5. Spin down cell debri, collect supernatant

6. Spin down phages overnight at 8000 rpm (12 hrs)

7. Re-suspend phages with gentle agitation at 4 degrees overnight

• Some bacteriophage give very robust yields from infection. In some cases, the lysate can be directly added to a cesium step gradient and a band will be visible, especially if you use a light in a dark room. Therefore, the overnight spin and resuspension described above are not necessary for most applications.

Making cesium stocks:

CsCl Stock:

Final density: Grams CsCl to add to 1mL buffer:

1.4 gm/mL: 0.610 gms to 1mL buffer

1.6 gm/mL: 1.01 gms to 1mL buffer

• Important: These are the amounts added to 1 mL, NOT to be added to a final volume of 1 mL

A quick way to ensure that your cesium concentration is correct is by simply calculating the density directly. Place a 100 μL pipette tip on a scale and tare it. With the same pipette tip, withdraw 100 μL of cesium solution and weigh it again. You can also use a refractometer to very accurately measure the amount of cesium in your solution.

Cesium gradient spin preparation:

We use Sw.41 rotor spinning at 35000 rpm for 2 hours. To add each successive concentration of cesium to the bottom of the tube, use a long (at least 4 inch) needle attached to a syringe. It is important that the needle diameter is thick enough that you can gently add the cesium without mixing the phases.

1. Add 10% sucrose in buffer to centrifuge tube. This amount will be variable, depending on how much sample needs to be added to completely fill the tube

2. Add 2ml of 1.4 gm/mL CsCl with syringe to tube below the 10% sucrose

3. Add 2ml of 1.6 gm/mL CsCl with syringe to tube below 1.4 gm/mL CsCl (*don’t let CsCl layers mix)

4. Add the phage sample carefully on top of the 10% sucrose

• Make sure the solution level is to the very top of the tube. If needed add more sucrose below the sample layer, or add buffer on top of the sample

5. Spin at 35000 rpm for 1.5 – 2 hours at 20 C

6. After completion of the spin, you should see a sharp off-white band where your 1.4 and 1.6 phases meet. These are your phage

7. Extract band carefully by puncturing through the side of the tube with a needle and syringe. Carefully suck the phage band out