How to run a qpcr plate on QuantStudio 6

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1 How to run a qpcr plate on QuantStudio 6 Important notes Check the compatibility of your mastermix with this instrument. In particular, mastermixes containing «high rox» concentrations should not be used. Rather choose the low rox version. Please ask us in case of doubt. If the concentration of rox in your mastermix is too high, you can solve that my unchecking the rox passive dye option during data analysis. How to start the run Wake-up the instrument from the touch screen (or switch on if needed). open the QuantStudio 6 and 7 Flex Real-Time PCR System software choose the New Experiment button -> From template : open the QS6 Flex folder and choose a template for 384-wells plate. We created an easy-to-use template called DontForgetToLoadPlate_QS6_384w_SYBRwithMelt_standardspeed_stdcurve_10ul.edt that you can use when you use the Hamilton robot. Otherwise, you can also choose a template that corresponds to your experiment, in the QS6Flex folder (taqman/sybr/etc, with/without melting curve, fast/standard run). In these templates, the volume in the well is 20 ul by default, therefore you ll have to change it afterwards.

2 choose the import option (at the top), and select the txt file created by the Hamilton robot, and click on start import" and answer yes : - Choose the relevant dye chemistry (TaqMan or SYBR), and Standard or Fast settings (choose Standard unless you have an assay and mastermix optimized for Fast settings): [2]

3 choose the Run method button on the left of the screen. if needed: choose 10 ul as Reaction Volume per Well, choose the number of cycles you want (40 by default). The melting (= dissociation) curve has been added automatically from the template (only relevant for SYBR green). Insert your plate inside the instrument. Click on the run button on the left of the screen: [3]

4 Click on start run, selecting the number appearing. Save your file in your own folder, and click again on the start run button if the run hasn t started yet (the button becomes red when the run has begun): After the run Analyzing the run with the correct passive reference - Choose the setup part on the left. Adapt the passive reference if needed (by default ROX is selected). Then, in the toolbar, choose analysis -> analyze. NB1: most of the time, a passive reference is included in the mastermix (often it s ROX). It is used to normalize for differences in volumes that can occur between wells. The passive reference is a fluorophore not involved in the qpcr reaction. The QuantStudio works well with low rox amounts. If your mastermix contains a high rox concentration, please unselect the passive reference option and reanalyze the data. NB2: if you don t know if your mastermix include ROX: Please check the Multicomponent plot under analysis (on the left). When you have a low ROX signal, it appears similar to this: [4]

5 - If you have a high ROX signal, it appears similar to this: [5]

6 - When you don t have ROX, it appears similar to this: Adapting the threshold and baseline The machine automatically adapts a threshold and baseline values. You should check for each gene that the threshold and baseline were correctly assessed. In the yellow tab under the amplification plot, under Target, choose your genes one by one in the dropping list, and check that the automatic threshold is in the linear part of the amplification curve. Adjust the baseline in case the beginning of the amplification curves look aberrant (rare case, ask us if needed). [6]

7 Exporting data - Data can be exported in excel format with the QS6 formatting, or with the formatting of other older instruments (options on top of the export window). Versions log - v1.05: many small changes as compared to initial draft, including analyses guidelines, and high/low rox recommendations. [7]

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