Biology Reference
In-Depth Information
Table 2
Standard cycling protocol for all ABI real-time
instruments
Thermal cycler protocol
Stage 1, Reps = 1
Step 1: Hold @ 95.0 °C for 10:00 (MM:SS)
Stage 2, Reps = 40 a
Step 1: Hold @ 95.0 °C for 00:10 (MM:SS)
Step 2: Hold @ 60.0 °C for 00:40 (MM:SS)
Dissociation protocol b
Stage 3, Reps = 1
Step 1: Hold @ 95.0 °C for 00:15 (MM:SS)
Step 2: Hold @ 60.0 °C for 00:30 (MM:SS)
Step 3: Hold @ 95.0 °C for 00:15 (MM:SS)
Settings
Sample Volume: 20 μ L 9,600 Emulation
Data Collection: Stage 2, Step 2 (60.0 @ 00:40)
a Default cycling program for a ABI qPCR machine is two-
step PCR (combined annealing/extension step @ 60.0 °C.
Make sure primers are designed suitably for two-step PCR
protocol
b Dissociation stage is required for SYBR green dye, and
instructed by the instrument manufacturer
4. Aliquot 18
μ
L reaction mix into each well of 96-well plate.
5. Add 2
L of templates, standard DNA or negative control,
mix by pipetting.
6. Seal the 96-well plate with adhesive fi lm.
7. Briefl y spin the plate to collect samples at the bottom of wells.
Check each well, and ensure no well has abnormal volume of
PCR reaction mix.
8. Turn on the real-time PCR machine, and setup up following
cycling protocol (Table 2 ).
9. Save the fi le before run, then click “Start” to initiate thermal
cycling.
10. When the run is fi nished, the data will be automatically added
and saved to the fi le in step 9 .
11. Go to tab “Results”, you can analyze data under subtab
“Amplifi cation Plot”, or view “Standard Curve” and
“Dissociation” reports.
12. Export and save subtab “Report” as *.csv fi le, which can be
open by Microsoft ® Excel.
μ
 
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