Difference between revisions of "MiR06"
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== Application in [[HRR]] == | |||
'''MiR06: Mitochondrial Respiration Medium - MiR06''' (MiR06 = MiR05 + Catalase). Total volume = 1 litre. | |||
<span style="color:#8B008B"> '''Caution:''' Chemicals stored in the fridge or freezer should be adjusted to room temperature before opening.</span> | |||
===Chemicals for the preparation of MiR05 stock solution=== | |||
::1) weigh given amounts of the listed chemicals (except K-lactobionate) and transfer to a 1000 ml glass beaker | |||
::2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily. | |||
::3) add ~800 ml H<sub>2</sub>O and dissolve the powder by stirring on a magnetic stirrer at ~30 °C | |||
::4) add 120 ml of [[#Preparation of K-lactobionate stock solution|K-lactobionate stock solution]] | |||
::5) adjust the pH to 7.1 with 5 N KOH at 30 °C | |||
::6) divide in to 20 ml portions and store at -20 °C in plastic vials | |||
{|border="1" cellpadding="5" cellspacing="0" | |||
|+ | |||
!Compound | |||
!Final conc. | |||
!FW | |||
!Addition to 1 litre final volume | |||
!Company, product code and storage | |||
|- | |||
|align="right"|EGTA | |||
|align="right"|0.5 mM | |||
|align="right"|380.4 | |||
|align="right"|0.190 g | |||
|align="right"|Sigma E 4378, 25 g, store at R.T. | |||
|- | |||
|align="right"|MgCl<sub>2</sub>.6H<sub>2</sub>0 | |||
|align="right"|3 mM | |||
|align="right"|203.3 | |||
|align="right"|0.610 g | |||
|align="right"|Scharlau MA 0036, 250 g, store at R.T. | |||
|- | |||
|align="right"|Taurine | |||
|align="right"|20 mM | |||
|align="right"|125.1 | |||
|align="right"|2.502 g | |||
|align="right"|Sigma T 0625, 25 g, store at R.T. | |||
|- | |||
|align="right"|KH<sub>2</sub>PO<sub>4</sub> | |||
|align="right"|10 mM | |||
|align="right"|136.1 | |||
|align="right"|1.361 g | |||
|align="right"|Merck 104873, 1000 g, store at R.T. | |||
|- | |||
|align="right"|HEPES | |||
|align="right"|20 mM | |||
|align="right"|238.3 | |||
|align="right"|4.77 g | |||
|align="right"|Sigma H 7523, 250 g, store at R.T. | |||
|- | |||
|align="right"|D-Sucrose | |||
|align="right"|110 mM | |||
|align="right"|342.3 | |||
|align="right"|37.65 g | |||
|align="right"|Roth, 4621.1, 1000 g, store at R.T. | |||
|- | |||
|align="right"|BSA essentially fatty acid free | |||
|align="right"|1 g/l | |||
|align="right"| | |||
|align="right"|1 g | |||
|align="right"|Sigma A 6003 fraction V, 25 g, store at 2-8 °C | |||
|- | |||
|align="right"|K-lactobionate | |||
|align="right"|60 mM | |||
|align="right"|358.3 | |||
|align="right"|120 ml of 0.5 M [[#Preparation of K-lactobionate stock solution|K-lactobionate stock]] | |||
|align="right"|Aldrich P 153516, 100 g, store at R.T. | |||
|- | |||
|} | |||
====Preparation of K-lactobionate stock solution==== | |||
::1) weigh 35.83 g K-lactobionate directly into a 250 ml glass beaker | |||
::2) add 100 ml H<sub>2</sub>O and dissolve by stirring on magnetic stirrer | |||
::2) check pH (is approx. around 2.0) and neutralize with [[#Preparation of 5 N KOH stock solution|5 N KOH]] | |||
::4) adjust final volume to 200 ml with H<sub>2</sub>O. It is best to use a 200 ml volumetric glass flask. | |||
::5) check pH again and adjust to 7 if necessary | |||
====Preparation of 5 N KOH stock solution==== | |||
'''KOH''' (Potassium hydroxide, ≥85% KOH basis, pellets); Aldrich P 1767, 1000 g, store at R.T.: FW = 56.11 | |||
__NOTOC__ |
Revision as of 10:34, 24 November 2010
Description
Mitochondrial respiration medium 06, developed for oxygraph incubations of mitochondrial preparations = MiR05 plus catalase.
Abbreviation: MiR06
Reference: MiPNet14.13; Gnaiger 2000 MitoInTheCold
Labels:
HRR: Chemicals; Media"Chemicals; Media" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property.
Application in HRR
MiR06: Mitochondrial Respiration Medium - MiR06 (MiR06 = MiR05 + Catalase). Total volume = 1 litre.
Caution: Chemicals stored in the fridge or freezer should be adjusted to room temperature before opening.
Chemicals for the preparation of MiR05 stock solution
- 1) weigh given amounts of the listed chemicals (except K-lactobionate) and transfer to a 1000 ml glass beaker
- 2) disrupt big lumps mechanically. It is recommended to do this before adding water, because during dissolution these lumps do not disintegrate easily.
- 3) add ~800 ml H2O and dissolve the powder by stirring on a magnetic stirrer at ~30 °C
- 4) add 120 ml of K-lactobionate stock solution
- 5) adjust the pH to 7.1 with 5 N KOH at 30 °C
- 6) divide in to 20 ml portions and store at -20 °C in plastic vials
Compound | Final conc. | FW | Addition to 1 litre final volume | Company, product code and storage |
---|---|---|---|---|
EGTA | 0.5 mM | 380.4 | 0.190 g | Sigma E 4378, 25 g, store at R.T. |
MgCl2.6H20 | 3 mM | 203.3 | 0.610 g | Scharlau MA 0036, 250 g, store at R.T. |
Taurine | 20 mM | 125.1 | 2.502 g | Sigma T 0625, 25 g, store at R.T. |
KH2PO4 | 10 mM | 136.1 | 1.361 g | Merck 104873, 1000 g, store at R.T. |
HEPES | 20 mM | 238.3 | 4.77 g | Sigma H 7523, 250 g, store at R.T. |
D-Sucrose | 110 mM | 342.3 | 37.65 g | Roth, 4621.1, 1000 g, store at R.T. |
BSA essentially fatty acid free | 1 g/l | 1 g | Sigma A 6003 fraction V, 25 g, store at 2-8 °C | |
K-lactobionate | 60 mM | 358.3 | 120 ml of 0.5 M K-lactobionate stock | Aldrich P 153516, 100 g, store at R.T. |
Preparation of K-lactobionate stock solution
- 1) weigh 35.83 g K-lactobionate directly into a 250 ml glass beaker
- 2) add 100 ml H2O and dissolve by stirring on magnetic stirrer
- 2) check pH (is approx. around 2.0) and neutralize with 5 N KOH
- 4) adjust final volume to 200 ml with H2O. It is best to use a 200 ml volumetric glass flask.
- 5) check pH again and adjust to 7 if necessary
Preparation of 5 N KOH stock solution
KOH (Potassium hydroxide, ≥85% KOH basis, pellets); Aldrich P 1767, 1000 g, store at R.T.: FW = 56.11