AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions
Jump to navigation
Jump to search
>Alan6017518 |
>Alan6017518 |
||
Line 17: | Line 17: | ||
*Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20 | *Aliquot reagent dye to 0.5mLtubes (2.2uL each tube) - Stor at -20 | ||
*Dilute 20X TE buffer to 1X working solution | *Dilute 20X TE buffer to 1X working solution | ||
*Take 1mL 20X TE buffer and dilute it with 19mL DNase-free water | *Take 1mL 20X TE buffer and dilute it with 19mL DNase-free water | ||
*Determine the volume of Working Reagent Required | *Determine the volume of Working Reagent Required |
Revision as of 15:16, 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
- 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
*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))
- 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: Prepare Oligonucleotide Standard
- Dilute the oligonucleotide standard provided(100ug/mL) to 100ng/mL with 1XTE buffer
- Load both samples and standard and into strip tubes
- The strip tubes 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: 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
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