AlanFung:Protocol/OliGreen ssDNA Stain: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Alan6017518
>Alan6017518
Line 22: Line 22:


==Step 2: Oligonucleotide Standard Curve==
==Step 2: Oligonucleotide Standard Curve==
*Dilute the oligonucleotide standard, provided at 100ug/mL to 50 fold in TE to make the 2ug/mL working solution
*Dilute the oligonucleotide standard provided(100ug/mL) to 2ug/mL  
*For low-range standard curve from 100pg/mL to 50ng/mL prepare a 20-fold dilution of the 2ug/mL oligonucleotide solution to yield a 100ng/mL oligonucleotide stock solution  
*For low-range standard curve, perform a 20-fold dilution to yield 100ng/mL oligonucleotide stock solution
*Mix well and incubate for 5 mins at RT, protected from light
*After incubation, measure the sample fluorescence using the fluorescence microplate reader and s
standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)
*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.


*Dilute 100ng/mL oligonucleotude stock solution
==Step 3==

Revision as of 21:06, 28 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
  • 96 well microplates
  • Microplate Reader

Overview

Procedures

Step 1: Preparing Reagent

  • Allow the reagent to warm to RT before opening the vial
  • Prepare an aqueous working solution of the Quant-iT OliGreen reagent by making a 200 fold dilution of the concentrated DMSO solution in 10nM Tris-HCL
1mM EDTA
pH 7.5 (TE)

Step 2: Oligonucleotide Standard Curve

  • Dilute the oligonucleotide standard provided(100ug/mL) to 2ug/mL
  • For low-range standard curve, perform a 20-fold dilution to yield 100ng/mL oligonucleotide stock solution
  • Mix well and incubate for 5 mins at RT, protected from light
  • After incubation, measure the sample fluorescence using the fluorescence microplate reader and s

standard fluorescein wavelength (excitation ~480nm, emmision ~520nm)

  • 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.

Step 3