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AlanFung:LabNotes/Probe Set Quantification/2009-6-3
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==Step 5: Sample Anaylysis== *Vortex all samples and reagent really well before loading *Add 25uL of the aqueous working solution of the Quant-T OliGreen reagent to each sample and standard *Load both samples and standard and into strip tubes *The strip tubes should look like this: [[Image:96 well plate.jpg]] *with the letters A-E (in A1-B2) being the standards and the numbers 1-11 (in C2-H4) being the samples *(it is read top-to-bottom then left-to-right; A1 β H1, A2 - H2, etc) *Mix well and incubate for 5 mins at RT, protected from light *After incubation, measure the sample fluorescence using the RT-PCR *Standard fluorescein wavelength (excitation ~480nm, emmision ~520nm) *Measure the absorbance at 520nm on the RT-PCR *For the Chromo4 Real Time PCR *Open the Opticon Monitor 3 program *Perform the following Incubate at 25C 4M 50S Incubate at 25C 10S Plate Read Go back to line 2 for one more time END *Subtract the fluorescnece value of the reagent blank from that of each of the samples *Use corrected data to generate a standard curve of fluorescence versus oligonucleotide concentration. *Record the ssDNA concentrations calculated by the worksheet *Incubate for 5 mins at room temperature, protected from light *Measure the fluorescence of the sample *Subtract the fluorescence value of the reagent blank from that of each of the samples *Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve
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