AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions
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*Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer | *Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer | ||
[[Image:OliGreen Standard.jpg]] | [[Image:OliGreen Standard.jpg]] | ||
==Step 3: Prepare Sample== | ==Step 3: Prepare Sample== |
Revision as of 15:47, 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
Overview
Procedures
Step 1: Preparing Working Reagent
*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
- Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer
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
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 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