User:Timothy L. Foley/Notebook/refolding matrix/2009/10/15

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Almost there

frikkin finally... it takes for ever to make these buffers... finished off the pH 6.5-9.2 buffer sets...

from my back of the envelope calculations, I determined that I would need to add 0.2 mL water to each tube to get to 9.5 mL total I did this.

other notes used 87% glycerol instead of 100%, so the folding reactions will be 17% glycerol instead of 20%

the amino acid solutions are 1/2 the original desired concentration (550), which makes them 275 mM.

We'll see what happens...

down to arraying these in 96 well plates

here's what we're gonna do:

array the solutions out in 800 uL whatman polypropylene (deep well) plates as storage plates for the buffer array...

they come a packs of 5, so i will set up 5 of them, but only add reducing agent and ligand to one, so it doesn't get burned out before it's time to shine...

the wells claim to hold 800 uL, but in reality, this volume leaves a large bubble on top (surface tension) so I figure a safe amount would be 600 uL (700 is just below the surface of the plate)... this gives, tentatively, 6 folding experiments from one stock plate... that can have reducing agents and ligands exchanged at will... ROCK!


to do this, I need to add:

534 uL each solution to each well, and then suppliment the entire plate with reducing agent (6 uL) and ligand (60 uL) before go time.

get this done.


this I can do. NB: care should be taken to vortex each sample before aliquoting, the glyerol does not mix well and makes a second dense phase on the bottom of the tubes.

the bufers were arrayed into Whatman 7701-5800 plates as described above. 1 pack of plates (5 total) were prepared, so there will be plenty to go around if this works and someone @Burkart wants to give it a shot...

the extra buffers (~ 5.5 mL in 15 mL Corning/falcon tubes) were placed in styrofoam tube racks (2 total) , wrapped copiously in saran wrap, sealed with a rigorous application of tape, and a label affixed to them explicitly stating "Buffers 1-50" and "Buffers 51-96".
tomorrow is go time in the AM. I will prepare the CoA & reducing agents, and get this mission going...and let it sit over the weekend @ 4*C to be assayed on monday.



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