-Shake until solutes have dissolved and add:
*250mM KCl 10 ml (for 1 L)or 2 ml (for 200 ml)
-AdjustpH to 7 with 5 N NaOH (~ 0.2 ml), or 1 N NaOH.
-Adjustvolume to 1L or 200ml with deionized H2O.
-Sterilizeby autoclaving for 20 min. at 15lb/sq. in. on liquid cycle.
-Allowmedium to cool to 600C or less and then add18 ml(for 1 L)or 3.6 ml(for 200 ml) of 20 % filter-sterilizedGlucose solution**.
-Add5 ml (for 1L) or 1 ml (for 200 ml) of a sterile solution of 2 M MgCl2***.
*250mMKCl: Dissolve 1.86g of KCl in 100ml of ddH2O.
**20%Glucose: Dissolve100 g of Glucose in 400 ml ddH2O. After the sugar has dissolved complete the volume to 500 ml and sterilizethrough a 0.22-micron filter.
***2M MgCl2: Dissolve19 g of MgCl2 in 90 ml ddH2O. Adjust volume to 100 ml with ddH2O and sterilize by autoclaving for 20 min. at 15 lb/sq. in. on liquid cycle. If you only have MgCl2 x 6H2O, then add 40.68 g ofthis to 90 ml ddH2O.
This was found according to the followingcalculations:
For 100 ml
MW: (Mg=24.30, Cl=35.45,H=1.01, O=16.00)
MgCl2 x 6H2O = 203.31 g/mol
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