1. Grow overnight culture of recipient strain.
2. Dilute donor P22 phage lysate in either T2 buffer or saline to allow MOI (Multiplicity of Infection) to be less than one except in cases to two particle transduction (tranducing marker is large). Since phage survives in T2 buffer longer than in saline, T2 buffer should be used when diluted phage is going to be kept.
3. If vitamin prototroph marker will be selected, the recipient cells ought to be washed by saline or minimal salt solution, then resuspended into minimal salt solution.
4. Take 0.1ml of the overnight culture, mix with 0.1ml of properly diluted donor P22 phage lysate, then:
a. if selective marker is Tcr, or prototrophic phenotype, cells and phages could be directly spreaded onto selective plates.
b. if selective marker is Knr, or Cmr, cells and phages could be spreaded onto NB plates and incubated at a proper temperature for overnight, then printed to selective plates. Alternatively, mixtures of cells and phage could be incubated at room temperature for one hour before being spreaded onto selective plates. Preincubation of NB plates gives rise to more transductants and more repeatable results.
c. if selective marker is Apr, mix of cells and phages could be directly spreaded onto selective plates, or preincubated in tubes for one hour, or preincubated on NB plates overnight before selecting Apr. Preincubation is recommennded.
d. if selection will be done on plates containing EGTA, cells and phages have to be mixed and preincubated at room temperature for one hour to allow infection finished.
e. If selected marker is GnR (gentamycin) use 20mg/ml gentamycin in LB (NB will not work!). Also, first spread transduction to an LB plate without drug and print to LB Gn after overnight incubation.
Incubate the plates at desired temperature until transductants grow up as colonies.
5. Nonselective marker could be screened either by directly printing to screen plates or by picking colonies to patch onto the same selective plates and then to replica print to screen plates.
6. Pick a few of each desires type of transductants from original selective plates or the master plates, streak out on green plates to grow for single colonies.
7. Pick phage-free colonies (white on green plate), streak cross over a line of P22 H5 phage lysate to test the phage sensitivity of the transductants.
8. Pick phage sensitive transductants, streak out on NB plates for single colonies.
9. Test phenotype of transductants by patching colonies onto a nonselective medium (e.g. NB plate) followed by replica-printing to a set of testing plates. All known phenotype of the strain have to be tested.
10. Take cells of a patch with correct phenotype from the master plate, streak out for single colonies on a NB plate.
11. To save the strain, see the section "Saving Strains".
Last Update: Thursday, 19-Jun-2014 12:09:48 PDT
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