Start new project 2 Find Rotor-Gene Shortcut on the desktop and open the software. New Run

Two rotors option 4 4) Choose the Rotor. There are two rotors available: 72 samples max, 10ul reaction volume - - It requires Qiagen Strip Tubes and Caps, 0.1 ml. To order 250 strips of 4 tubes and caps for 1000 reactions use Qiagen Order##981103 -$162.00 OR or 36 samples, 20ul reaction volume, requires PCR Tubes, 0.2 ml - 1000 thin-walled flat cap tubes for 1000 reactions Qiagen Order##981005 - $98.20. 5) Make sure locking ring is attached and secured.

Samples layout 6 6) For your convenience choose default setting A01-A08

PCR temperature Edit Profile settings 8 7) Choose detector 8) Edit Profile, do not Edit Gain

Holding -95°C-10min; Cycling – 95°C-15sec; 60°C-1min; 40Cycles Melting - default 9) Choose the length of initial denaturation step – 10 min recommended. 10) You have an option to add 50°C-2min holding step before denaturation step allowing optional treatment with UNG to eliminate carryover of PCR products from previous reactions – ABI-TaqMan kit #4304437 Only (Ten-minute incubation at 95 °C is necessary to cleave the dU-containing PCR product generated in the low temperature (18 to 50°C) incubation, to substantially reduce AmpErase UNG activity, and to denature the native DNA in the experimental sample. Because UNG is not completely deactivated during the 95 °C incubation, it is important to keep the reaction temperatures greater than 55 °C, to prevent amplicon degradation).

Editing PCR cycling settings 11) Highlight the step to edit

Editing PCR cycling settings Note, acquiring data is set at annealing/extension step

Editing PCR cycling settings 11 11) You can add additional annealing step 55-60°C -15-30sec; keep reaction temperatures greater than 55 °C.

Editing data Acquisition settings You have a choice of various commonly used dyes: FAM=SYBR Cy5 JOE~VIC See next slide for reference. Note, ROX is not being used by RotorGene as Passive Reference. Presence of ROX in reaction doesn’t interfere with settings.

Dyes reference VIC Ref: http://194.209.195.227/351.0.html

Editing PCR cycling settings 13 12 12) Cycling – set for 40 cycles. 13) Do not edit Melt 14) OK done with cycle editing 14

Calibration 15 15) Click to calibrate – only Project is new

Calibration – cont’d Fluorescence detection sensitivity 16 17 16) Click to perform Auto calibration before first acquisition, tube position A01. 17) make sure you have placed your samples in the rotor position A01 (you can Edit position if you like)

Calibration – cont’d 18 18) You can add/remove Channels

Save and start 19 19) Click Next to Save Profile and Start your Run

Save and start 20 20) click Start

Save and start 21) Save your profile when prompted

Save and finish 21 21) Click Finish – Instrument will start the run

Run in progress 22) You can observe the progress by looking at current temperature

File saved

DATA ANALYSIS 21 21) Open your file for analysis

DATA ANALYSIS, cont’d 22 23 22) Click Analysis, highlight cycling A FAM – if SYBR (single acquisition, or A+B-if using two channels) 23) Click Show

DATA ANALYSIS, cont’d 24 24) Follow the prompts to manually set up a Threshold line

ANALYSIS – THRESHOLD LINE 26 25) Click Threshold manual set up button 26) NOW CAREFULLY SELECT WHERE YOUR THRESHOLD SHOULD BE - Single click on the curves window will set up at threshold line. Once Threshold is set, Ct values calculated. 25

Melting point analysis 27 27) Melting point analysis, click Melt, then Show

Reports 28 Observe your melting curves calculated for you 28) Go to reports to retrieve and save the data

Ct values data 29 29) Highlight Cycling A.FAM /Quantitation (Concise)/ Show

Ct values data Save your Ct values report and proceed to Melting point report

Melting point data 30 30) Highlight Melt A.FAM /Melt Full report/ Show

Melting point data Save your melting point data