AlanFung:LabNotes/Probe Set Quantification/2009-6-3: Difference between revisions

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*Subtract the fluorescence value of the reagent blank from that of each of the samples
*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
*Determine the oligonucleotide concentration of the sample from the standard curve generated in Oligonucleotide Standard Curve
==Data==
[[Image:OliGreen Test 06_03_09.jpg]]
==Discussion==
The data looks quite reasonable, but still some of the data points doesn't make sense
==Improvements==
*Perform 3 replicate for each concentration
*Add in the OliGreen dye in the dark and prepare the reaction as quick as possible

Latest revision as of 18:11, 3 June 2009

Probe Preparation[edit]

Objective[edit]

  • Perform only the high-range standard curve

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

File:High Range OliGreen Standard.jpg


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:

File: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

Data[edit]

File:OliGreen Test 06 03 09.jpg

Discussion[edit]

The data looks quite reasonable, but still some of the data points doesn't make sense

Improvements[edit]

  • Perform 3 replicate for each concentration
  • Add in the OliGreen dye in the dark and prepare the reaction as quick as possible