Tim Burland (Last Update May 10, 1995)
(Use to grow axenic amoebae & plasmodia in liquid)
To 900mL double-distilled or distilled-deionized water, add:
Adjust pH to 4.6 with KOH, autoclave 15lb for 15min, store away from light at room temp.
Before use, add 10ml hemin solution per litre.
For plates, mix equal volumes of molten 2.5% agar and [SDM + hemin] immediately before pouring plates.
Essential additive for SDM
Make molten 1.5% agar by autoclaving 15g DifCo Bacto agar in each litre of double-distilled or distilled-deionized water
To each litre of molten agar, add 65ml SDM and mix. Pour plates immediately—do not let the molten agarose stand at elevated temperature, lest the low pH hydrolyse the agar, resulting in sloppy plates.
Use for growing amoebae at high density on lawns of bacteria on plates; recipe developed by Roger Anderson
To 1000 mL double-distilled or distilled-deionized water, add
Autoclave 20min 15lb/sq in., cool to safe temperature, Pour plates.
Use for Physarum spore platings and for recloning amoebae on bacterial lawns
Needed to make DBL plates
For diluting or washing axenic amoeba
To 900 mL double-distilled or distilled-deionized water, add
Dissolve solids, pH to 5.0 with KOH, make to 1000ml, sterilize by autoclaving.
Use for making spherules from plasmodia
To 1000 mL double-distilled or distilled-deionized water, add in this order:
Adjust pH to 3.8 with KOH. Autoclave to sterilize.
Nuclear Homogenization Solution
250mM Sucrose / 0.1% Triton-X100 / 15mM CaCl2 / 10mM Tris pH 7.5
Store frozen or make fresh from sterile stocks of :
1M sucrose, 10% Triton-X100, 1M 15mM CaCl2 and 1M Tris pH7.5
For lysing isolated nuclei
100mM EDTA / 20mM Tris pH8 / 0.5% SDS. Store room temp.
Just before use, add 1/20th vol. Proteinase K stock
Dissolve ribonuclase A at 1mg/mL and ribonuclease T1 at 1000u/mL in 10mM potassium acetate pH5.2.
Heat to 80o for 10min to inactivate DNAses, then store -20o
10mM Tris pH8, 1mM EDTA. Autoclave to sterilize.
To grow bacteria
To 800ml distilled water add
Dissolve solids, pH to 7.4 with NaOH, make to 1000ml with distilled water, autoclave 20min 15lb/sq in.
After autoclaving, add 5ml 20% glucose.
To grow lotsa bacteria
To 800ml distilled water add
Dissolve solids, pH to 7.4 with NaOH, make to 1000ml with distilled water, autoclave 20min 15lb/sq in.
After autoclaving, add 5ml 20% glucose.
To grow bacteria
Add 15g DifCo Bacto Agar per litre of LB broth before autoclaving.
40mM Ultra-Pure Sucrose (GibCo-BRL Cat#5503UA)
10mM Hepes pH 8.2 (GibCo-BRL Cat#845-1344IL)
Filter-sterilize, store room temp.
Supplied in the Promega Luciferase Assay Kit Cat #E1500:
10mM EDTA, 100mM DTT, 50% glycerol, 5% Triton X-100, 125mM Tris, adjusted to pH7.8 with H3PO4
For lysis of Physarum amoebae, dilute the buffer to 1x with water containing the following protease inhibitors:
The 1x solution with protease inhibitors can be stored for several weeks at -20° in 1-mL aliquots; thaw on ice just before use. The stocks of protease inhibitors should be stored at -70°.
The Promega kit provides lyophilised luciferase substrate and assay buffer; mixing the two generates the assay reagent, which should be stored in 100-500-uL portions at -70° immediately after mixing until ready to use.