Setting up the instrument for PCR (SYBR and TAQMAN)
Base on ABI 7900
Book instrument
Go to ‘www.mrl.ims.cam.ac.uk/intranet/booking/genomics/’
Clk the instrument you want
384 = ABI 7900
96 = ABI 7300
This takes you to the Google Calender
Log in
Click on the calendar slot you want
Enter name and edit the time slot
Start programe
Clk ‘SDS 2.3’
Clk ‘Okay’ at the user identification page
Select the following
Assay: Standard Curve (AQ) absolute quantification
Cond: 384 Well Plate
Template: Blank Template
Clk ‘Okay’
Select the detectors to use
Clk ‘Tools’ then ‘Detector Manager’
Select the detector you want to use
TAQMAN
Reporter = FAM
Quencher = TAMRA
SYBR
Reporter = SYBR
Quencher = NA
Clk ‘Add to plate document’: You can add more detectors if we use different
detectors simultaneously
Clk ‘Done’
Assign the detector to the wells you want
Select the wells by dragging the mouse
Assign the detector to use on the right hand pane
Check that passive reference = ROX
Instrument setup
Clk ‘Instrument’ tab
Clk ‘Thermal Cycler’
Check that
Mode = Standard
Sample volume = usually 12ul
The cycling parameters are as above
9600 emulation is OFF
For SYBR runs you need to add dissociation stage; this checks the purity of the
amplification. If >1 product is amplified, there will be 2 peaks
Clk the figure at the end of ‘Stage 3’
Clk ‘Add dissociation stage’ button at the bottom of the menu
Connect to instrument and starting the run
Clk ‘Real time’ Tab
Clk ‘Connect to instrument’
Clk ‘Open/Close’
Load the plate in
Clk ‘Open/Close’
Clk ‘Start Run’
Analysing the output
Open SDS2.3
Clk ‘File’
For ABI 7900; D:/Applied_Biosystems/SDS Documents/Chong/xxx
Select the wells you want to analyse
Clk ►; 2 new tabs will appear ‘Result’ and ‘QC summary’
Look at the left pane
Green colour = okay
Yellow with numbers = error; 1 = empty wells/none detected
Under the ‘Result’ tab, select ‘Amplification plot’
Look also at the results table on the bottom left
Optimising the output
Target
CT values should be between 25 to 35
For 18s, the CT are usually very low because of high concentration of 18s
The CT cutoff line (the green line) should be optimized
Optimum cutoff should be across the most linear phase of the amplification
The CT cutoff is different between different genes; each gene should be
optimized separately
The CT cutoff line should aim to avoid areas of noise
Optimising the CT cutoff
Select all the wells with the same gene
Exclude empty wells and –rna wells
Analyse the data
If CT cutoff not optimize; Clk ‘Analysis Setup’
Adjust the baseline region; eg CT between 3 to 25. This tells the software
where the baseline extend to
Manual adjust allows you to manually move the line on the plot
Export the results
Clk ‘File’
Clk ‘Export’
Clk ‘Result table’
Clk ‘Selected wells’
Format SDS2.3; tick group by replicates
Select location to save
Disconnect instrument