Quantitative PCR uses fluorescent dye to quantify the relative difference in sequence copy number between two samples. The dye used is SYBR Green which binds to dsDNA but not to ssDNA, when comparing two qPCR reactions of a given sequence SYBR Green's signal is proportional to sequence copy number.
The Bitan lab does not have a qPCR machine. There is one in the de Vellis lab on the third floor that you can use if you get permission from Cristina Ghiani (cghiani at mednet stop ucla stop edu). You must also sign up to use the machine at least a day in advance of your experiment using the calendar that hangs over the machine.
NOTE: The thermal cycler _MUST_ be turned on at least 20 minutes prior to starting the qPCR reaction in order to allow the bulb to warmup. This is extremely important for the upkeep of the machine and the reproducibility of your data.
Bio-Rad iQ SYBR Green Supermix Manual
The manual for this kit is here.
Total volume per well is 25 μL.
12.5 μL iQ SYBR Green Supermix
100-500 nM each primer
1 μL sample
ddH2O to 25 μL
You need a qPCR plate for this, it should have conical wells and it should be specially coated so it doesn't interact with protein.
Run the MyiQ program from the Desktop.
From the "View Protocol" tab choose "Create a New Protocol."
In the "Show Options" box check the box for "Melt Curve."
In addition to editing incubation times and temperatures you can also insert and delete cycles or steps with the green buttons at the bottom of the screen.
When you are satisfied with your protocol save it and choose "Run With Selected Plate Setup."
This is a sample protocol for an ALISA experiment, it's saved as C:\Program Files\Bio-Rad\MyiQ\User1\Andrew\Andrew.tmo.
|Cycle||Repeats||Step||Dwell Time||Setpoint||Melt Curve||Temp+||Temp-|
Understanding your data
When the experiment is over MyiQ will show you a figure full of sigmoidal lines. The x-axis is cycle number and the y-axis is fluorescence. You can think of the y-value as being proportional to the number of copies of the DNA sequence present in the reaction.
Cycle-threshold: A fluorescence threshold will be calculated by MyiQ for your experiment. The number of cycles it takes a sample to cross this threshold is its cycle-threshold (Ct). You can compare relative copy number between two samples by comparing their Ct values. Let ΔCt be the difference between the two Ct values. The following assumes 100% replication efficiency at each cycle:
- if ΔCt = 0 the samples started the experiment with equal copy number.
- if ΔCt = 2 one sample started with 2x the copy number of the other sample.
- if ΔCt = 4 one sample started with 4x the copy number of the other sample.
MyiQ gives you some control of the value of the fluorescence threshold (use the mouse to drag the threshold around). In general you would like to keep the threshold as low as possible while keeping it above background noise, this will tend to minimize variability between replicates and improve reproducibility.