Making Heads From 9-amN110/13-am/ C2ts30 Lysogens in 7136


We produce heads using the lysogen to reduce the amount of background phage (there is no input phage which one will need to be concerned with). The repressor is on at low temperature, thus no phage are produced; at high temperature the repressor is off and the phage are produced. Because the genotype is 9-(am N110) no tails are made (thus only heads) and it is 13-(amH101): the lysis is delayed. So the cells fill with heads.



Grow cells which contains the phage as a lysogen at 30oC to about 4x10E8 cell/ml in super broth.


Shift the cells to 42oC till the temperature reaches 39oC. Add equal volume of 57oC broth; the repressor will come off and the phage will enter the lytic cycle. Once the cells have reached the 39oC shift to 39oC to continue the head production.


Grow 2-3hrs. Ice. Concentrate the cells. Lysis by CHCl3 and Freeze / Thawing.


Pellet debris, resuspend the pellet and treat again to lysis, pellet debris. Combine the supernatants (heads should be here) and pellets.


Next: Plate +/ - tails of both the supernatant and pellet to determine the plaque forming units (pfu).


Pellet Heads: 90 min. 15 K in ss34 (or 35 K 1hr in ultra rotor)


Resuspend in high Mg2+ (50 - 100 mM) in a Tris or PO4 Buffer to help stabilize the DNA in the heads. Try not to be above 1x10E12 heads/ml as there may not be enough Mg2+ to stabilize the DNA and this may pop the heads. If the prep is very viscous (high amt of DNA) you have lost the infectious heads due to popping.


For ultimate in high-purity heads: CsCl gradient