AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions

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==Procedures==
==Procedures==
==Step 1: Preparing Reagent==
==Step 1: Preparing Working Reagent==
*Allow the reagent to warm to RT before opening the vial
*Allow the reagent to warm to RT before opening the vial
*Determine the volume of Working Reagent Required
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL))
*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
*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
  1mM EDTA

Revision as of 22:27, 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 Working Reagent

  • Allow the reagent to warm to RT before opening the vial
  • Determine the volume of Working Reagent Required
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL))
  • 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

File:OliGreen Standard.jpg

  • Load both standard and samples into the 96 well plate
  • The 96 well plate should look like this:

File: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 – H1, A2 - H2, etc)
  • Mix well and incubate for 5 mins at RT, protected from light
  • After incubation, measure the sample fluorescence using the fluorescence microplate reader
  • 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.


  • Measure the absorbance at 520nm on a plate reader
  • For the PFBH biotech core facility plate reader:
  • Turn on the plate reader (a switch in the back of the machine)
  • Open the SOFTmax Pro program
  • Click Setup, a window will pop up
  • Set the excitation wavelength to 480nm
  • Set the emission wavelength to 520nm
  • Click “Read”, a window will pop up
  • Select “Normal”, then click “OK”
  • The tray will enter the machine and after a few seconds it will come back out and a chart of numbers will appear in the program
  • Click “File”, “Import/Export”, “Export…”
  • Choose the location of the USB memory stick (or Floppy disk) and click “Save”
  • Analyze absorbance of standards (known concentrations) and samples (unknown concentrations) to determine concentrations of samples
  • Copy the chart of the absorbance readings (from the outputted text document) into the appropriate space in the worksheet provided
  • Record the ssDNA concentrations calculated by the worksheet

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