AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions

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>Alan6017518
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*Subtract the fluorescnece value of the reagent blank from that of each of the samples
*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.
*Use corrected data to generate a standard curve of fluorescence versus oligonucleotide concentration.
1.Measure the absorbance at or near 562 nm on a plate reader
 
i.For the PFBH biotech core facility plate reader:
 
a.Turn on the plate reader (a switch in the back of the machine)
*Measure the absorbance at 520nm on a plate reader
b.Open the SOFTmax Pro program
*For the PFBH biotech core facility plate reader:
c.Click Setup, a window will pop up
*Turn on the plate reader (a switch in the back of the machine)
d.Set the single wavelength to 560 nm and click “OK”
*Open the SOFTmax Pro program
e.Click “Read”, a window will pop up
*Click Setup, a window will pop up
f.Select “Normal”, then click “OK”
*Set the excitation wavelength to 480nm
g.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
*Set the emission wavelength to 520nm
h.Click “File”, “Import/Export”, “Export…”
*Click “Read”, a window will pop up
i.Choose the location of the USB memory stick (or Floppy disk) and click “Save”
*Select “Normal”, then click “OK”
2.Analyze absorbance of standards (known concentrations) and samples (unknown concentrations) to determine concentrations of samples and the volume required for 10 μg of protein
*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
i.See Appendix B for the Excel worksheet which will do this analysis
*Click “File”, “Import/Export”, “Export…”
ii.Copy the chart of the absorbance readings (from the outputted text document) into the appropriate space in the Excel worksheet
*Choose the location of the USB memory stick (or Floppy disk) and click “Save”
iii.Record the protein concentrations calculated by the Excel worksheet (should be around 2 μg/μL, but may vary significantly)
*Analyze absorbance of standards (known concentrations) and samples (unknown concentrations) to determine concentrations of samples
iv.Record the protein  volumes calculated by the Excel worksheet (should be around 5 μL, but may vary significantly; not to exceed 20 μL)
*Copy the chart of the absorbance readings (from the outputted text document) into the appropriate space in the worksheet provided
v.Record the buffer volumes calculated by the Excel worksheet (should be 1/3 of the protein volume, to dilute 4X to 1X in protein)
 
*Record the ssDNA concentrations calculated by the worksheet


==Step 3: Sample Anaylysis==
==Step 3: Sample Anaylysis==

Revision as of 21:36, 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-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)
  • 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.


  • 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