Setup and analysis QuantStudio 5 Real-Time PCR System

Transcription

1 Setup and analysis QuantStudio 5 Real-Time PCR System Applied Biosystems

2 Software 1 Open software Click on the QuantStudio Design and Analysis Software icon to start the software 2

3 Software 2 Home window 2. From the home window you can reach the different steps to start and analyse and programme an experiment over the tool bars or the submenus. Create a new experiment or open existing experiment 2. Setup of a new experiment 3

4 2. Settings 2.1 New experiment 2. Click on Create New Experiment [1] icon to start a completely new experiment, or Template [2] to start an experiment with previously defined setup 4

5 2. Settings 2.2 Properties Enter in Name [1] the name of your run file (YYYYMMDD-name) and in User name [2] your shortcut. Click Next [3] to get to the next page. 5

6 2. Settings 2.3 Method Enter your reaction volume per well [1] (25µl for liquid and 15µl for lyo kits) and add/modify different stages/temperatures/times based on the Fast-track mastermix protocol [2]. To create a template press Save [3] and save as template (edt. file). 6

7 2. Settings 2.4 Plate 2. In the Quick Setup [1] choose Passive Reference None [2] in the field Plate Attribute. 7

8 2. Settings 2.4 Plate 9. To define targets and samples add new target [1] and enter a target name [2]. Choose colour for respective target [3]. Select task [4a/b] (unknown, standard, NC) and add the quantity in the empty field next to task if selected standard. Add new sample [5] and enter the sample name [6]. Choose the wells in the plate view [7] to include respective samples and targets. It is possible to create a library of all targets present in FTD assays. Click on Action [8] and save the predefined target to library [9]. 8

9 2. Settings 2.4 Plate To choose targets from library, click on Import from Library [1]. Search for your targets with the filter options Target [2] and begins with [3] and the predefined FTD kit shortcuts [4] and press Apply Filter [5]. Underline your desired targets and transfer them to your target name list by clicking Add Selected [6]. 9

10 2. Settings 2.5 Run 2. To begin the run click the Start Run [1] button. All cyclers connected with the computer will be shown as serial number [2]. Choose the right serial number and you will be asked to save the file and the run starts. 10

11 2. Settings 2.5 Run You can interrupt the run at any time by pushing Stop run [1]. The estimated remaining run time is also displayed [2]. 11

12 3. Analysis 3.1 Results After the run is completed you are automatically directed to Results. By default you can see the Amplification plot logarithmic graph type. You can open runs also from the home menu and open multiple plates simultaneously. 12

13 3. Analysis 3.2 First settings Click on the Eye icon and change the settings according to your needs [1]: Graph from Logarithmic to Linear type [2] Plot colour from Well to Target [3] Change Threshold Auto and Auto Baseline [4] to individual adjusted values Validate your choice by pushing Analyse [5] 13

14 3. Analysis Baseline/Threshold 2a. 3b. 3a. 2b. Choose your target [1] and unmark Threshold Auto [2a]. To set the threshold either insert the threshold value or adjust the threshold line [2b] with the cursor. The start and end of the baseline have to be changed manually the green and red arrows [3a] after unmarking Auto Baseline [3b]. Validate your choice after each step by pushing Analyze. 14

15 3. Analysis Baseline/Threshold You can also open Analysis settings [1] to adjust threshold and baseline. Underline the desired targets and unmark Default Settings, Automatic Threshold and Automatic Baseline. Set threshold and baseline [2] for your targets and Apply [3] your settings. 15

16 3. Analysis Baseline/Threshold In Advanced Settings [1] you can adjust the threshold for single wells. Underline the desired well and target and continue as described before. 16

17 3. Analysis 3.3 Standard curve In Standard Curve Settings [1] it is also possible to save standard curves [2] or upload [3] standard curves from previous runs. 17

18 3. Analysis Results You can find the determined Ct values either in each well of the Plate Layout [1] or sorted in a table in the Well Table [2]. The quantities can be found in the Well Table next to the Ct value [3], to see the quantity in the Plate Layout move the cursor on the respective well [4]. 18

19 3. Analysis Standard results To verify your standard results change the view to Standard Curve [1]. Choose your desired target [2] to see Slope value, Y-intercept, R2 value and amplification efficiency (Eff%) [3]. 19

20 If you have any further questions or suggestions, please contact us at or check out the FAQ section on our website