"Bochner" Selection of TetS



Collective Lab Wisdom, ca. 1990
       Flask A
       12  g    agar
        4  g    tryptone
        4  g    Yeast Extract
       50 mg    chlorTet
      400 ml    water

       Flask B
        8  g    NaCl
        8  g    NaH2PO4.H2O
      400 ml    water
autoclave 20'
cool to pouring temp.

add:
      9.6 mg  fusaric acid 
             (light-sensitive; 
              dissolve with a drop of NaOH solution, 
              or in dimetyl-formamide; 
              important that it's really dissolved)
      4 ml    20mM ZnCl2
mix and pour
use immediately (within two days, 12 hours is best)

Two methods

1. Plate 0.1 ml per 106/ml for Tn10's (for Tn10d-tet, use 0.2 ml o/n).

2. Drop of liquid, streak (can use independent cultures).

(TetR are tiny, TetS are about 3X as large, i.e., everyone grows)

Important tips

1. Autoclave chlorTet with tryptone
2. Don't autoclave fusaric acid
3. Don't autclave ZnC2 with media
4. Test at different dilutions of cells for method 1

Cute tricks

1. TetS will grow as tinys on NB+0.1xTet (2mg/ml);
--Try streaking putatives on NB+0.1xTet (2 mg/ml)

2. Different phenotypes on green plates with 0.6 mg/ml Tet (subtle)
--TetR darker green, sometimes larger
--TetS lighter green, with dark point in center

Kelly says:

Age of the plates is critical
Use fresh plates
42°C => clean: 30°C => lawn -- temp is important!
Also: Can screen on minimal - but can't do initial selection

Barry says:

Grow fresh colonies on a rich Tet plate,
pick to rich Tet media,
grow to just late log;
TetR is very induced and plates are cleaner.

Minimal "Bochner" plates
(I'm not sure whose trick this is, but these work for initial selection. ECK)
       Flask A
        15   g    agar
        50  mg    chlorTet
       500  ml    water

       Flask B
10 g NaH2PO4.H2O 2 g K2HPO4 2 g NH4Cl 0.1 g MgSO4 (7H2O) or 0.4 ml 1M stock 500 ml water
autoclave 20' cool

add:
        1  ml     2 mg/ml fusaric acid
        5  ml     20 mM ZnCl2
        4  ml     50% glucose
        (+ any needed supplements to identify auxotrophs)

Last Update: Thursday, 19-Jun-2014 11:59:51 PDT
This page has been viewed 68 times.
Eric Kofoid eckofoid at ucdavis.edu