Hi Nagarjun,

To see if your mutant phage produces infectious phage, I'd grow a broth culture from a colony of your transformed cells, doing the same for the wild-type M13KO7 in parallel as a control. As the phage confers kanamycin resistance, you need to keep kanamycin present while growing your cultures. Scaling up your culture during log phase will allow you to generate more phage, but probably isn't necessary for detection. The phage can be concentrated from the media using a double PEG precipitation procedure (there are lots of protocols available on the web). The resultant precipitate can be analysed on western blot with an anti-M13 antibody (ideally not an anti-pVIII monoclonal, as the epitope might be disrupted by your mutation). Your WT control should tell you if your culture conditions and precipitation are working, and indicate if your mutation impairs production of infectious particles.

Best wishes

Dear Nagarjun,

The M13 KO7 phage has an origin of replication derived from p15A inserted into the normal M13 origin. While the phage genome is not packaged as efficiently as a phagmid containing a wild-type f1 or M13 origin, in the absence of the phagmid viable phage particles containing the helper genome should be generated by your XL1-Blue cells. As long as your modification to gVIII does not prevent assembly of new virus particles, your transformed culture of XL1-Blue should express phage into the media that can be easily purified by precipitation with 8000 MW PEG/NaCl.

Hope this helps.

Dear Alexander,

Thank you very much for your reply and information.

So If I culture the transformed XL1-Blue cells, they should produce phage into the media, but it can't be sure that my modification to gene VIII is viable to produce phage. So I need to do a single colony culture once I reach the OD of 0.6, I need to transfer it larger volume right. Do I need to add the kanamycin/ tetracycline to my culture or not.  

Hi Nagarjun,

To see if your mutant phage produces infectious phage, I'd grow a broth culture from a colony of your transformed cells, doing the same for the wild-type M13KO7 in parallel as a control. As the phage confers kanamycin resistance, you need to keep kanamycin present while growing your cultures. Scaling up your culture during log phase will allow you to generate more phage, but probably isn't necessary for detection. The phage can be concentrated from the media using a double PEG precipitation procedure (there are lots of protocols available on the web). The resultant precipitate can be analysed on western blot with an anti-M13 antibody (ideally not an anti-pVIII monoclonal, as the epitope might be disrupted by your mutation). Your WT control should tell you if your culture conditions and precipitation are working, and indicate if your mutation impairs production of infectious particles.

Best wishes

Thank very much for the detailed information.