User:Brigette D. Black/Notebook/Brigettes Notebook/2009/10/31/Further-ly Optimized Bulk Ideas

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Try maybe two or three times as many MTs (4.213 uM) and the same concentration of kinesin (1: 12.6 ratio).

  • Gilbert:
    • 12uM tubulin
    • 0.2 uM Kinesin
  • Last Assay:
    • 1.51 uM tubulin
    • .33 uM kinesin

Last time, 3 uL produced (1.0666*10^-9) mols Pi which implies that the same number of moles of ATP were used per sample, or about (2.666 *10^-8) mols for the entire system. (2.666*10^-8 mols/ 75*10^-6 L = .356 mM used). To be on the safe side, let's use at least 2mM ATP.

Idea: Super PEM-T (2.37x as much taxol as regular PEM-T). Why? I have realized that diluting the reactions too much has lead to nothing but heartache and bad data. So if I am going to use more MTs, I don't want to end up over-diluting the reaction. Also, if I simply add 2.37 times as many MTs, I will have to add about 2.37x as much of everything else to accommodate for the volume change. So, less diluted, less kinesin, more rocking.

MT+ ATP concoction

  • (3uL each, make 25 uL extra total)
    • Total Volume needed: 100 uL
  • ATP :
    • 10uL (one aliquot) => 10 mM ATP (seems well over what is needed but still over what I think the malachite green + spectrophotometer can resolve well).
  • BME: 3 uL
  • MTs: 87 uL MTs (5 uL tubulin + 82 uL Super PEM)
    • 5.22 uM "Stock"
    • 4.514 uM in solution
    • Total volume: 100 uL

Save 25 uL to measure autohydrolysis. To this 25 uL, add 1.67 uL PEM (to keep concentrations the same as that in the reaction). 3 uL to 37 uL PEM in cuvette, develop and measure normally. (Makes 4 duplicated points)

Reaction

  • 70 uL MT+ATP (4.213 uM MTs + 9.333 mM ATP + 2.8 uL BME)
  • 5 uL kinesin (0.333 uM)
  • (ATP Background: 0.7 mM)

Add 3 uL reaction to 37 uL PEM. Develop and measure as usual. Makes 12 duplicated measurements.

Time Ideas

  • 0 min MTs+ATP
  • 30 min MTs+ATP
  • 60 min MTs+ATP
  • 90 min MTs+ATP
  • 0 min Reaction
  • 2 min Reaction
  • 4 min Reaction
  • 6 min Reaction
  • 10 min Reaction
  • 15 min Reaction
  • 20 min Reaction
  • 30 min Reaction
  • 45 min Reaction
  • 60 min Reaction
  • 75 min Reaction
  • 90 min Reaction

SUPER PEM-T

  • 2.37 uL 100 uM taxol per mL (make 1 mL to be safe). Add 997.6 uL PEM.
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