AlanFung:Protocol/OliGreen ssDNA Stain

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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.

Step 3