User:Brigette D. Black/Notebook/Brigettes Notebook/2009/07/24/NanoDrop! And standard curves

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The Nanodrop came in! My first act of duty was to make a standard curve using the nanodrop, just to see how it compares with the one in Osinski's lab. I made 11 total cuvettes with the following concentration and volume of 1mM phosphate.

  • Cuvette 1: Blank (0mM Pi)
  • Cuvette 2: 25 uM (1uL)
  • Cuvette 3: 50uM (2uL)
  • Cuvette 4: 100uM (4uL)
  • Cuvette 5: 150uM (6uL)
  • Cuvette 6: 250uM (10uL)
  • Cuvette 7: 375uM (15uL)
  • Cuvette 8: 500uM (20uL)
  • Cuvette 9: 625uM (25uL)
  • Cuvette 10: 750uM (30uL)
  • Cuvette 11: 1mM (40uL)

To these cuvettes I added DI water to bring the total volume to 40uL per cuvette. I then added 200 uL of perchloric acid (quencer). Then added 200 uL of Malachite Green solution. I waited ten minutes for each cuvette and then read out the absorbance at 650 nm.

  • Cuvette 1: Blank
  • Cuvette 2: 0.044
  • Cuvette 3: 0.093
  • Cuvette 4: 0.191
  • Cuvette 5: 0.334
  • Cuvette 6: 0.498
  • Cuvette 7: 0.717
  • Cuvette 8: 0.805
  • Cuvette 9: 1.01
  • Cuvette 10: 0.948
  • Cuvette 11: 1.017

I measured these values again at 14 minutes to get a sense of how time sensitive the malachite green really is. The absorption at 14 minutes was measured as follows.

  • Cuvette 1: Blank
  • Cuvette 2: 0.08
  • Cuvette 3: 0.145
  • Cuvette 4: 0.245
  • Cuvette 5: 0.402
  • Cuvette 6: 0.608
  • Cuvette 7: 0.843
  • Cuvette 8: 0.945
  • Cuvette 9: 1.194
  • Cuvette 10: 1.09
  • Cuvette 11: 1.225

Image:Nanostandard.png

As before, the linear fit is not that great. There is an average error between the line and the measurements of 25%, but it is well enough to approximate the concentration of phosphate from the absorbance. Also, the data gathered today (at least for low concentrations) is very similar to the previous standard curve that was make in the other lab, It does seem at this point that the nanodrop is not incredibly accurate at high concentrations (greater than 500 uM). However, we won't be needing to measure concentration quite that high, so I think we are safe

Image:Standardcomparison.jpg

Lower Concentration Standard Curve

As previous measurements showed, we should expect low concentrations of phosphate when we use kinesin and microtubules. I thought that it would be a good idea to make a standard curve that ranged from 0 - 50 uM Pi so that I could better estimate the activity levels when that time comes around again.

  • Cuvette 1: Blank
  • Cuvette 2: 12.5 uM (0.5uL)
  • Cuvette 3: 18.75 uM (0.75uL)
  • Cuvette 4: 25 uM (1uL)
  • Cuvette 5: 31.25 uM (1.25 uL)
  • Cuvette 6: 37.5 uM (1.5 uL)
  • Cuvette 7: 43.75 uM (1.75 uL)
  • Cuvette 8: 50 uM (2uL)

The absorbances were measured this time with a bucket on top of the nanodrop. Since the cuvette holder is not light tight, I thought that it would be best for now and in the future to make the all of the measurements at least partially shielded from ambient light. Also, I have seen a few papers/protocols that measure the absorbance of a malachite green assay at 660 nm instead of 650. Since this machine can make several wavelength measurements in very little time, I thought I'd try at both wavelengths and see which gave the larger reading. In the list below, the readings at 660 nm are in parenthesis.

  • Cuvette 1: Blank
  • Cuvette 2: 0.025 (0.024)
  • Cuvette 3: 0.026 (0.025)
  • Cuvette 4: 0.037 (0.035)
  • Cuvette 5: 0.065 (0.062)
  • Cuvette 6: 0.070 (0.068)
  • Cuvette 7: 0.084 (0.083)
  • Cuvette 8: 0.090 (0.089)

So the measurements at 660nm are just slightly less than those at 650 nm. Below is a plot of the standard curve for low concentrations, which will hopefully come in handy very soon!

Image:Lowconcentrationstandard.png

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