Editing
AlanFung:LabNotes/Probe Set Quantification/2009-6-2
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
='''Probe Preparation'''= ==Objective== *Perform only the high-range standard curve ==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 ΞΌL)) 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 β 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
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information