AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions

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==Step 2: Oligonucleotide Standard Curve==
==Step 2: Oligonucleotide Standard Curve==
*The 96 well plate should look like this:
[[Image:96 well plate.jpg]]
[[Image:96 well plate.jpg]]
*with the letters A-I (in A1-B3) being the standards and the numbers 1-15 (in C3-H6) being the samples
*(it is read top-to-bottom then left-to-right; A1 – H1, A2 - H2, etc)
*Dilute the oligonucleotide standard provided(100ug/mL) to 2ug/mL  
*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
*For low-range standard curve, perform a 20-fold dilution to yield 100ng/mL oligonucleotide stock solution

Revision as of 21:20, 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

  • The 96 well plate should look like this:

File:96 well plate.jpg

  • with the letters A-I (in A1-B3) being the standards and the numbers 1-15 (in C3-H6) being the samples
  • (it is read top-to-bottom then left-to-right; A1 – H1, A2 - H2, etc)
  • 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: Sample Anaylysis

  • Dilute your sample solution in TE to a final volume of 1mL
  • Add 200uL of the aqueous working solution of the Quant-T OliGreen reagent to each sample
  • 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