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
- The 96 well plate should look like this:
- 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