User talk:James Chappell/ Sandbox
Discussion: Threshold of Visualisation
Vincent 06:08, 25 September 2007 (EDT): Hi, I understand that you are trying to reduce the financial impact of using cell-extract by doing your tests in water. However, aren't you worried that your results, on the visual threshold, might be difficult to use as the cell extract has a significant fluorescent background ? Same for the degradation rate. The cell extract is a very different environment. What do you think ?
Yeah it is true that the threshold is likely to be affected by the cell extract. We know for the calibration curve and degradation rate we have to use cell extracts, but thought we could avoid it with this test. However, your right about the cell extract having an affect. I think we could carry out this test to reduce the range of where the threshold is and then knowing this test with cell extract.
If we carry out the threshold first then, we can define it in water and then can test in cell extract. I think the threshold will be reduced as emitted light from DsRed is more likely to absorbed.
- Vincent 04:56, 26 September 2007 (EDT): It might also be that the background fluorescence of the cell extract could mask the fluorescence from DsRed. So, at the end you would require a higher concentration of DsRed to make it visible.
One thing i think worth testing is the background fluorescence of DsRed using the red filter in the fluorometer.
- Vincent 04:56, 26 September 2007 (EDT): you mean the cell extract, instead of DsRed, no ?
Also we are going to look into trying to find a suitable replacement for cell extract, e.g. media and premix. With this it would help reduce cell extracts used for calibration curve for dsRed + GFP. We are planning to do a quick test tomorrow with a few types.
- Vincent 04:56, 26 September 2007 (EDT): Why do you want a calibration curve between GFP and DsRed ?