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

2. 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## $ OR or 36 samples, 20ul reaction volume, requires PCR Tubes, 0.2 ml thin-walled flat cap tubes for 1000 reactions Qiagen Order## $98.20.
5) Make sure locking ring is attached and secured.

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

4. PCR temperature Edit Profile settings8
7) Choose detector
8) Edit Profile, do not Edit Gain

5. 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 # 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).

6. Editing PCR cycling settings
11) Highlight the step to edit

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

8. Editing PCR cycling settings11
11) You can add additional annealing step 55-60°C sec; keep reaction temperatures greater than 55 °C.

9. 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.

10. Dyes reference
VIC
Ref:

11. Editing PCR cycling settings13 12
12) Cycling – set for 40 cycles.
13) Do not edit Melt
14) OK done with cycle editing 14

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

13. Calibration – cont’d Fluorescence detection sensitivity16 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)

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

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

16. Save and start 20
20) click Start

17. Save and start
21) Save your profile when prompted

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

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

20. File saved

21. DATA ANALYSIS 21
21) Open your file for analysis

22. 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

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

24. ANALYSIS – THRESHOLD LINE26
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

25. Melting point analysis27
27) Melting point analysis, click Melt, then Show

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

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

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

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

30. Melting point data
Save your melting point data