Studier Lysate Prep

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Summary

How to make a lysate from a plaque preparation. We also use this protocol for preparation of a quick stock from previously made lysate prep.

Protocol

1. Add 5mL fresh overnight (BL21 if wild-type T7) into 15 mL of T broth (125 mL flask).
   • I often use LB --Sri Kosuri 15:18, 3 Jun 2005 (EDT)
2. Add a single plaque to above (or a drop of lysate)
3. Shake at 30°C until lysis is visually apparent (2-3hr)
4. As soon as lysis is observed and appears to be complete, add 1g of NaCL to the flask and dissolve by shaking.
   • Adding NaCl prevents phage particles from getting pulled down during centrifugation.
5. Once NaCl is dissolved, centrifuge for 10 min at 10,000 rpm at 4°C.
   • Studier has observed that leaving released phage in the cell debris causes a reduction in the kinetics of phage binding (personal communication). It becomes difficult to get good time courses in this case. This can often happen even over the course of an hour. So it is important to catch lysis and spinning down the debris as soon as possible. It is recommended when making a large stock that will be used in timing experiments, that you check that most of your phage adsorb to the cell in the first 30 seconds.
6. Store tube in fridge away from light (usually good indefinitely, e.g., retains absorption well).

References

1. Studier FW. The genetics and physiology of bacteriophage T7. Virology. 1969 Nov;39(3):562-74. DOI:10.1016/0042-6822(69)90104-4 |