AlanFung:LabNotes/Probe Set Quantification/2009-6-11

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Revision as of 20:35, 11 June 2009 by >Sam Chiang (New page: ='''Probe Preparation'''= ==Objective== *Perform only the high-range standard curve *Add OliGreen Dye in complete darkness *Perform 3 replicate for each concentration *Add in the OliGreen ...)
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Probe Preparation

Objective

  • Perform only the high-range standard curve
  • Add OliGreen Dye in complete darkness
  • Perform 3 replicate for each concentration
  • Add in the OliGreen dye in the dark and prepare the reaction as quick as possible
  • Test the accuracy of the standard with Re-Test DNA

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

File:High Range OliGreen Standard.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:

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

Discussion

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

Improvements