Talk:MiPNet14.13 Medium-MiR06

Preparation of MiR05 (MiR06) stock solution

• This is the version up to 2013-09-07:

1. Weigh given amounts of the listed chemicals (except BSA and lactobionic acid) 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 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. Transfer the MiR05 stock solution to a 1000 ml volumentric glass flask and add H20 to a final volume of 1000 ml = MiR05.
7. Check pH again and adjust if necessary with small amounts of 5 N KOH.
8. Dissolve the BSA in a subsample of the MiR05 stock solution and add to the final MiR05 (the separate preparation of the BSA solution is recommended, since BSA produces foams that do not dissolve easily).
9. To prepare MiR06, add 280 000 units of catalase (100 mg of catalase powder containing 2800 U/mg solid) per 1 L MiR05 (280 units / ml final concentration).
10. Divide into 40 ml portions and store at -20 °C in plastic vials .

MiR06 can also be prepared by adding 5 µl of the catalase stock solution directly into the O2k-chamber filled with MiR05 at the start of the experiment. The final catalase concentration in the 2 ml Oxygraph-2k chamber = 280 U/ml.

Ready-made powder

For a high-altitude project in Bolivia, MiR06 had to be shipped as a complete mixture of dry powder, and be made up with deionized H₂O and pH-adjusted on-site. Respiratory results were excellent in permeabilized muscle fibres with complex SUIT protocols. In contrast, BIOPS was non-functional when prepared from the mixture of dry powder. --Gnaiger Erich 20:35, 3 August 2012 (CEST)

Cost

Q: For calculating the running costs: what are the typical costs for MiR05 per run?

A: For a general discussion of running costs see »Low O2k running costs. We once calculated the chemical costs for MiR05 to be about 60 € /liter (excluding VAT). Calculated with 3 ml/per chamber (for a 2 ml chamber) this would amount to about 0.4 € per run (2 chambers). Calculation is based on Austrian prices; and on the assumption that no "left overs" are taken into acount, i.e. the entire package is used; not taken into account are work, water, KOH for adjusting the pH ..

Fasching Mario 12:07, 13 October 2011 (CEST)

Inhibitory effects

• Lack of ADP stimulation with either glutamate&malate or succinate&rotenone excludes an explanation by rotenone retention effects.
• Do you observe the problems when using mitochondrial respiration medium (MiR05 or MiR06 with permeabilized cells), but not when using cell culture medium (intact cells)? If yes, then there could be an inhibitory compound in MiR05/MiR06. In one lab, MiR05 had to prepare always fresh: After freezing, there was an inhibition or respiration. In general, MiR05 and MiR06 (including MiR05Cr and MiR06 Cr) are perfectly OK after freezing. There must have been something in the stock chemicals that caused an inhibitory effect after freezing. Perhaps you can get a batch of medium form another lab, just to try if this medium might solve the problem?

--Gnaiger Erich 20:00, 8 November 2011 (CET)

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