AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions
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==Step 2: Oligonucleotide Standard Curve== | ==Step 2: Oligonucleotide Standard Curve== | ||
*Dilute the oligonucleotide standard | *Dilute the oligonucleotide standard provided(100ug/mL) to 2ug/mL | ||
*For low-range standard curve | *For low-range standard curve, perform a 20-fold dilution to yield 100ng/mL oligonucleotide stock solution | ||
*Mix well and incubate for 5 mins at RT, protected from light | |||
*After incubation, measure the sample fluorescence using the fluorescence microplate reader and s | |||
standard fluorescein wavelength (excitation ~480nm, emmision ~520nm) | |||
*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. | |||
==Step 3== |
Revision as of 21:06, 28 May 2009
Probe Preparation
Samples & Materials
- Quant-iT OliGreen ss DNA Assay Kit (Cat. no. O11492)
*Quant-iT OliGreen ss DNA Reagent (Component A), solution indimethylsulfoxide (DMSO) *20X TE Buffer (Component B) *Oligonucleotide Standard (Component C) 18-base M13 sequence primer, with the sequence 5'-TGTAAAACGACGGCCAGT-3'
- Sterile, distilled, DNase-free water
- 96 well microplates
- Microplate Reader
Overview
Procedures
Step 1: Preparing Reagent
- Allow the reagent to warm to RT before opening the vial
- Prepare an aqueous working solution of the Quant-iT OliGreen reagent by making a 200 fold dilution of the concentrated DMSO solution in 10nM Tris-HCL
1mM EDTA pH 7.5 (TE)
Step 2: Oligonucleotide Standard Curve
- Dilute the oligonucleotide standard provided(100ug/mL) to 2ug/mL
- For low-range standard curve, perform a 20-fold dilution to yield 100ng/mL oligonucleotide stock solution
- Mix well and incubate for 5 mins at RT, protected from light
- After incubation, measure the sample fluorescence using the fluorescence microplate reader and s
standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
- 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.