AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions

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   with the sequence 5'-TGTAAAACGACGGCCAGT-3'
   with the sequence 5'-TGTAAAACGACGGCCAGT-3'
*Sterile, distilled, DNase-free water
*Sterile, distilled, DNase-free water
*96 well microplates
*Microplate Reader


==Overview==
==Overview==
Line 15: Line 13:


==Procedures==
==Procedures==
==Step 1: Preparing Reagent==
==Step 1: Preparing Working Reagent==
*First Time
*Allow the reagent to warm to RT before opening the vial
*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
*Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20C
  1mM EDTA
  *First Time
  pH 7.5 (TE)
*Dilute 20X TE buffer to 1X working solution
  *Take 1mL 20X TE buffer and dilute it with 19mL DNase-free water


==Step 2: Oligonucleotide Standard Curve==
*Determine the volume of Working Reagent Required
*The 96 well plate should look like this:  
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL))
One tube of aliquot gives you 440uL of OliGreen dye
 
==Step 2: Prepare Oligonucleotide Standard==
*Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer
[[Image:OliGreen Standard.jpg]]
 
==Step 3: Prepare Sample==
*Dilute sample to range of 50pg/mL to 50ng/mL
*Typical probes concentration ranges from 4-20ng/uL
*Perform a dilute according to the Table
[[Image:OliGreen Sample.jpg]]
 
==Step 4: Prepare OliGreen Reagent==
*Prepare an aqueous working solution of the Quant-iT OliGreen reagent by making a 200 fold dilution of the concentrated DMSO solution in 1XTE Buffer
*Add 437.8uL of 1X TE Buffer to the 2.2uL dye aliquot
 
==Step 5: Sample Anaylysis==
*Vortex all samples and reagent really well before loading
*Add 25uL of the aqueous working solution of the Quant-T OliGreen reagent to each sample and standard
*Load both samples and standard and into strip tubes
*The strip tubes should look like this:  
[[Image:96 well plate.jpg]]
[[Image: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
*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)
*(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
*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
*After incubation, measure the sample fluorescence using the RT-PCR
standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
*Standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
 
*Measure the absorbance at 520nm on the RT-PCR
*For the Chromo4 Real Time PCR
*Open the Opticon Monitor 3 program
*Perform the following
Incubate at 25C 4M 50S
Incubate at 25C 10S
Plate Read
Go back to line 2 for one more time
END
*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
*Record the ssDNA concentrations calculated by the worksheet
i.For the PFBH biotech core facility plate reader:
a.Turn on the plate reader (a switch in the back of the machine)
b.Open the SOFTmax Pro program
c.Click Setup, a window will pop up
d.Set the single wavelength to 560 nm and click “OK”
e.Click “Read”, a window will pop up
f.Select “Normal”, then click “OK”
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
h.Click “File”, “Import/Export”, “Export…”
i.Choose the location of the USB memory stick (or Floppy disk) and click “Save”
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
i.See Appendix B for the Excel worksheet which will do this analysis
ii.Copy the chart of the absorbance readings (from the outputted text document) into the appropriate space in the Excel worksheet
iii.Record the protein concentrations calculated by the Excel worksheet (should be around 2 μg/μL, but may vary significantly)
iv.Record the protein  volumes calculated by the Excel worksheet (should be around 5 μL, but may vary significantly; not to exceed 20 μL)
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)


==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
*Incubate for 5 mins at room temperature, protected from light
*Measure the fluorescence of the sample
*Measure the fluorescence of the sample
*Subtract the fluorescence value of the reagent blank from that of each of the samples
*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
*Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve

Latest revision as of 22:12, 31 May 2009

Probe Preparation[edit]

Samples & Materials[edit]

  • 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

Overview[edit]

Procedures[edit]

Step 1: Preparing Working Reagent[edit]

*First Time
  • Allow the reagent to warm to RT before opening the vial
  • Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20C
*First Time
  • Dilute 20X TE buffer to 1X working solution
*Take 1mL 20X TE buffer and dilute it with 19mL DNase-free water
  • Determine the volume of Working Reagent Required
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL))
One tube of aliquot gives you 440uL of OliGreen dye

Step 2: Prepare Oligonucleotide Standard[edit]

  • Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer

File:OliGreen Standard.jpg

Step 3: Prepare Sample[edit]

  • Dilute sample to range of 50pg/mL to 50ng/mL
  • Typical probes concentration ranges from 4-20ng/uL
  • Perform a dilute according to the Table

File:OliGreen Sample.jpg

Step 4: Prepare OliGreen Reagent[edit]

  • Prepare an aqueous working solution of the Quant-iT OliGreen reagent by making a 200 fold dilution of the concentrated DMSO solution in 1XTE Buffer
  • Add 437.8uL of 1X TE Buffer to the 2.2uL dye aliquot

Step 5: Sample Anaylysis[edit]

  • Vortex all samples and reagent really well before loading
  • Add 25uL of the aqueous working solution of the Quant-T OliGreen reagent to each sample and standard
  • Load both samples and standard and into strip tubes
  • The strip tubes 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 RT-PCR
  • Standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
  • Measure the absorbance at 520nm on the RT-PCR
  • For the Chromo4 Real Time PCR
  • Open the Opticon Monitor 3 program
  • Perform the following
Incubate at 25C 4M 50S
Incubate at 25C 10S
Plate Read
Go back to line 2 for one more time
END
  • 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.
  • Record the ssDNA concentrations calculated by the worksheet
  • 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