Similar Protocol
From fresh O/N colonies:
1. innoculate 3 mls 2xYT + antibiotic by loop, toothpick, pipet tip...
2. add 1 ul of helper phage
3. incubate 1 hr., 37 C, shaking
4. add kanamycin
5. grow to saturation, but not more than ~48 hours
From not-so-fresh colonies:
1. innoculate 3 mls 2xYT
2. incubate 3-4 cell cycles, or ~1-1.5 hrs., 37 C, shaking
3. add antibiotic
4. incubate 20 min., 37 C, shaking
5. add 1 ul of helper phage
6. incubate 1 hr, 37 C, shaking
7. add kanamycin
8. grow to saturation, but not more than ~48 hours
Using a colony: Inoculate 3 ml LB* w/ colony, add
1 ul M13K07 Helper Phage, and shake for 1 hour.
Add 3 ul Kanamycin, shake for 24-48 hours.
Spin down cells and save supernatant.
Using a culture: Inoculate 3 ml LB* w/ 10-30 ul overnight culture, add
1 ul M13K07 Helper Phage, and shake for 1 hour.
Add 3 ul Kanamycin, shake for 24-48 hours.
Spin down cells and save supernatant.
*LB should contain Carb and Tet, or appropriate antibiotic. colony and culture should be fresh (O/N)