AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions
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==Step 1: Preparing Working 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 | ||
*Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20 | |||
*Dilute 20X TE buffer to 1X working solution | |||
*Determine the volume of Working Reagent Required | *Determine the volume of Working Reagent Required | ||
(# standards (5) + # samples) x (replicates (2)) x (volume per well (100 μL)) | (# 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 | *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 2: Oligonucleotide Standard Curve== | ==Step 2: Oligonucleotide Standard Curve== |
Revision as of 00:14, 31 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
- Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20
- Dilute 20X TE buffer to 1X working solution
- 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 1XTE Buffer
- Add 437.8uL of 1X TE Buffer to the 2.2uL dye aliquot
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
- Load both standard and samples into the 96 well plate
- 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)
- 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 program
- 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
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