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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


high-resolution terminology - matching measurements at high-resolution


MiR06

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