litic phage contamination during panning! - (May/31/2007 )

Hi!!
I am working with phage display system using Fab library and i have after 3 round of panning a LETAL contamination from Litic Phage. Could someone help me in somehow???? HELP!!!!

---

hi.

can you give a bit more detailed information & how did you foundout that you have contamination?

---

I did three round of panning using an immobilized antigen, after each elution with acid solution, i infected XL1blue cells with my output phage, and after i did the superinfection with the helper phage. After the superinfection a let them grow for 2 h before adding kanamicine and then i let them grow overnight but there was no grow after the third round!!!! The culture was almost clear.

---

[/quote]

I did three round of panning using an immobilized antigen, after each elution with acid solution, i infected XL1blue cells with my output phage, and after i did the superinfection with the helper phage. After the superinfection a let them grow for 2 h before adding kanamicine and then i let them grow overnight but there was no grow after the third round!!!! The culture was almost clear.
[/quote]

Sometime the cells used to amplify phages maybe too old or no longer susceptable to infection which mostly depends of how the stock culture was prepared. To verify this you need to check the input titres if no colonies are obtained that may indicate that cells are the cause on drop or loss on enrichment observed after round 3.

---