AlanFung:Lab Notebook/Probe Set Quantification: Difference between revisions

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='''Probe Preparation'''=
*[[AlanFung:Probe_Set_Quantification/OliGreen|OliGreen]]
==Objective==
*To optimize condition for the quantification of ssDNA using OliGreen
*Using Re-Test ssDNA as a standard sample
 
==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
[[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 23:40, 1 June 2009