Making Protein in the Phage system

CHP - last updated: January 29, 1999

Step 1

• add 10 ml 7136 su- overnight to 1L of LB or 1L of super at 30⁰C, grow to mid log (3-6 hours)

  This strain will not suppress the ambers so the protein or structure of interest will accumulate.

• Grow to ~ 4 x 108 cells/ml counting under the microscope 320cells /16 boxes or 20 cells /1 box.

Step 2 

Before infection, take a sample of the culture examine under the microscope. The cells should look like sausages and should be swimming.

• Cell killing control - cells that are infected do not make a colonies, therefor ones needs to plate cells before infection determine the live cells and then 10 min. after infection for surviving bacteria

T=0 plate for colonies at 10^-6 dilution 0.1 ml.

Step 3

• infect culture with high titer phage stock at an m.o.i. (multiplicity of infection) of 5-10 phage/cell. ( Vol of media * cell count * 5 / phage titer)

  Phage are so small in comparison to the cell this should give at least 1 phage/cell while others may get 10 phage/cell

Step 4
Plate for bacterial survival Dilute 10-4 0.1 for colonies

Step 5
Grow for 3 hours at 30⁰C (or other more permissible temperature)

Step 6
Before harvesting, take a few mls

• Examine under the scope. Cells should appear long (filled with product), not small and round (in stationary)
• Add a few drops of CHCl3 it should lysis.

If the cells are small and do not lysis there is a problem. Consider aborting the prep.

Step 7
Spin culture - in centrifuge bottles - at 5K rpm for 5 minutes 4K/20 mins RC2C +

Step 8
resuspend pellet in buffer B with 40mM octoglucoside.

• If a phage prep use the Tris /Mg buffer.

Step 9
Can begin freeze/thaw cycles of purification procedure