Lidstrom:MM1 recipe

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About MM1

• MM1 is used by some methylotrophs.
• It is made of a a sodium & potassium phosphate buffer with magnesium, ammonium, and trace elements added.
• Helen Chan and Janet Matsen tested whether the two solutions that are autoclaved separately can be mixed before autoclaving 2/2015.
  • We tested MM1 liquid and MM1 agarose media.
  • For the liquid MM1, solutions that are autoclaved before mixing did fine whether the mixing of solutions happened before or after autoclaving. Some small precipitates formed the day after, but they dissolved within a few days. Note that in that experiment, trace elements had not been added.
  • When a similar test of mixing was done for MM1A solutions, the solution that was mixed before it was autoclaved was more brown.
  • We are currently validating whether SIP3-4 grows fine on media prepared both ways (mixed before autoclaving & mixed after autoclaving).
• The trace element solution includes EDTA to help keep the ions in solution and prevent precipitation.
  • Don't filter trace elements or any solution that contains iron, says Frances 2/2015.

Trace Elements Recipe & Tips

Tip: trace elements seem to need to be pH adjusted to allow for dissolving for the ZnSO4/EDTA mix and prevent precipitation. Helen Chan found that pH adjusting the EDTA + ZnSO4 solution to ~pH 6 allowed the EDTA and ZnSO4 to dissolve and prevented precipitation after addition of Solution 2.

• Total volume: 1L (adjust accordingly)
  • Solution #1 (500 mL ddH2O):
    • 4.4 g ZnSO4 (Heptahydrate)
    • 10 g EDTA*Na2 (Dihydrous)
  • Solution #2 (500 mL ddH2O):
    • 1.466 g CaCl2 (Dihydrate)
    • 1.012 g MnCl2 (Tetrahydrate)
    • 0.22 g (NH4)6Mo7O24 (Tetrahydrate)
    • 0.314 g CuSO4 (Pentahydrate)
    • 0.322 g CoCl2 (Hexahydrate)
    • 0.998 g FeSO4 (Heptahydrous)
• Procedure:

1. Prepare solution #1 mixture (in a 1L bottle) according to these criteria:
   1. Add EDTA*Na2 slowly, stirring with a stir bar while you do so.
   2. After EDTA*Na2 has been well dissolved (little to no cloudiness), add ZnSO4 scoop by scoop.
      1. For every 3 scoops, add 200 uL of 10 M KOH. This solution must not be incredibly acidic in order for each component to be soluble.
   3. You should end with a pH of 6. It would be optimal to make this solution with a pH probe attached.
2. Prepare solution #2 mixture (in a 1L bottle) and add a stir bar to assure that all components are dissolved well.
   1. Do not let this solution sit long before autoclaving. This solution should be light pink/orange by the time you combine it with solution #1.
3. Before autoclaving, combine bottles into a 2L bottle.
4. Autoclave as soon as possible (within ~ 1 hr) or else the solutions begin to precipitate.
5. The Lidstrom Lab has historically kept the final solution in the refrigerator.

MM1 liquid recipe

• Total volume: 2L (adjust accordingly)
  • Solution #1 (1L ddH2O):
    • 5.06 g K2HPO4 (Anhydrous)
    • 5.18 g NaH2PO4 (Monohydrous)
  • Solution #2 (1L ddH2O):
    • 0.4 g MgSO4 (Heptahydrous)
    • 1 g (NH4)2SO4 (Anhydrous)
• Procedure:

1. Mix constituents of solution #1 and #2 separately in 2L bottles.
   1. Currently considering the effects of mixing #1 & #2 before autoclaving.
2. Organize tiny bottles to be filled with media post autoclave.
   1. Each bottle should eventually contain ~150 mL of media.
3. Autoclave clean the solution-containing bottles along with capped empty bottles to aliquot the final solution into.
4. Add 2 mL Visnaic trace elements into bottle #2.
5. Pour equal amounts of solution #1 and #2 into the smaller bottles.

MM1A recipe

• Total volume: 2L (adjust accordingly)
  • Solution #1 (1L ddH2O):
    • 5.06 g K2HPO4 (Anhydrous)
    • 5.18 g NaH2PO4 (Monohydrous)
  • Solution #2 (1L ddH2O):
    • 0.4 g MgSO4 (Heptahydrous)
    • 1 g (NH4)2SO4 (Anhydrous)
    • 30 g Bacto Agar
• Procedure:

1. Mix constituents of solution #1 and #2 separately in 2L bottles.
   1. Currently considering the effects of mixing #1 & #2 before autoclaving.
2. Organize tiny bottles to be filled with media post autoclave.
   1. Each bottle should contain ~150 mL of media.
3. Autoclave large bottles and tiny bottles.
4. Add 2 mL Visnaic trace elements into bottle #2.
5. Pour equal amounts of solution #1 and #2 into the smaller bottles.
6. Let cool until solidification for storage.
   1. If you wish to pour plates immediately after autoclaving, place bottles into 50C incubator before pouring into plates.