1. Measure RNA via NanoDrop to determine volume of 1 ug.
2. cDNA Synthesis:
3. qPCR with SensiFast SYBR Kit
Sign up to reserve Roche Real Time Cycler 1 via microsoft outlook.
Switch on lightcycler to preheat (room A114).
Start lightcycler480 software (Login: Kapahi, Pass: Roche480)
denature: 1: None
PCR cycles: 40: Quantification
melt: 1: Melting Curves
cool: 1: None
Target: 95; Acquisition Mode: None; Hold: 00:02:00; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 95; Acquisition Mode: None; Hold: 00:00:05; Ramp Rate: 4.8; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 60; Acquisition Mode: Single; Hold: 00:00:20; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0
Target: 95; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 4.8
Target: 40; Acquisition Mode: None; Hold: 00:01:00; Ramp Rate: 2.5
Target: 65; Acquisition Mode: None; Hold: 00:00:01; Ramp Rate: 1
Target: 95; Acquisition Mode: Continusous; Ramp Rate: 0.02; Aquisitions: 25
Target: 40; Acquisition Mode: None; Hold: 00:00:10; Ramp Rate: 2.5; Sec Target: 0; Step Size: 0; Step Delay: 0
create new experiment from template (SensiFast SYBR)
Save Experiment (floppy disk symbol)
Use 4titude white plate (BASIC Scientific 4ti-0381) on cooling plate on ice.
Dilute primer pairs to 5 uM per single primre (1:20, if stock 100 uM) in RNase-free water.
Dilute cDNA reaction with RNase-free water to have sufficient amounts
Add 3 ul cDNA to 4titude plate.
Prepare master mix for each primer pair
5 ul SYBR-Mix
2 ul Primer pair
3 ul diluted cDNA
= 10 ul q PCR reaction
Add 7 ul of master mix to appropriate wells. Reaction volume per well is 10 ul.
Close plate with adhesive film (VWR 60941-078)
Spin plate shortly
Insert plate into lightcycler and start run (~55min)
4. Analyze qPCR with Lightcycler software
Press analyze button (method: Abs Quant/2nd derivative)
Press calculate button after selecting the right wells
In sample window: right click to export data table
In amplification window: right click to export chart as jpeg
Press analyze button (method: Tm calling)
In melting peaks window: right click to export chart as jpeg
Save data and close application