AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions
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>Alan6017518 |
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*20X TE Buffer (Component B) | *20X TE Buffer (Component B) | ||
*Oligonucleotide Standard (Component C) 18-base M13 sequence primer, | *Oligonucleotide Standard (Component C) 18-base M13 sequence primer, | ||
with the sequence 5'-TGTAAAACGACGGCCAGT-3' | |||
*Sterile, distilled, DNase-free water | *Sterile, distilled, DNase-free water | ||
== | ==Overview== | ||
Allow | |||
==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 | |||
[[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]] | |||
*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 |
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
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
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:
- 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