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now: Wed Oct 18 21:28:48 2017 ... mod: Tue Jul 2 00:14:36 2013
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HoechstFluorimetry
HoechstFluorimetry
Using a DNA-fluorescent dye like Hoechst 33258 can greatly increase the sensitivity at which DNA concentrations can be quantified.

Mix equal volumes of DNA in water with 2x Hoechst 33258 staining solution. Measure in a UV-fluorometer.

Make a standard curve with the following final DNA concentrations:

The Hoechst fluorescence has a reasonably linear response to DNA concentrations up to about 1000 ng/ml.

2x
Hoechst 33258 staining solution
final concentration
200 μl
1M Tris pH 8.0
20 mM
40 μl
500 mM EDTA pH 8.0
2 mM
4000 μl
1M NaCl
400 mM
4 μl
1 mg/ml Hoechst 33258
400 ng/ml
5756 μl
H2O
10 ml final total volume

Hoechst 33258 fluorescence is sensitive to salt concentration. A final concentration of 200 mM minimizes variability.

Stellar work there everyone. I'll keep on raeding.
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