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Create Protocol
*Log into Thermo Cloud ([https://apps.thermofisher.com/apps/dashboard/#/ https://apps.thermofisher.com/apps/dashboard] using the login information for your lab (the Bridges Lab account data is located on the side of the machine.)
*Click on the '''File Folder icon''' and find your folder (or the folder in which you saved your run).
*Select the file with extension '''.eds'''. The program will automatically take you to the '''Results''' tab of your run.
*Above the graph, use the dropdown box to select '''Melt Curve'''.
*Click on '''Target''' above the plate schematic to select one target at a time.
**Verify that there is only one (prominent) peak for each of your targets.
**Verify that each of your replicates amplified for each target.
**If there is a target that yields 2+ peaks over all samples, mark that target as unusable in the '''Primer Database'''.
**Exclude samples that did not the above criteria by clicking the well not the plate schematic and '''Omit''' under the '''Action''' dropdown box above the plate schematic.
*Check the technical replicates by choosing the '''Table View''' and arranging them according to target. Verify that the '''CT''' values have <1.5 difference. Omit outliers.
*Click '''Next''' and '''Export''' your results to the appropriate project folder.
*Under '''App Connect''' on the right hand side click on '''Relative Quantification''' (qPCR). Open your run.
*Under the '''Overview''' tab, assign biological groups to each of your samples (if needed).
*If you need to omit samples from the analysis, do so under the '''Data Review''' tab by clicking on the affected target, highlighting, and omitting the wells with outliers.
*Under the '''Analysis''' tab click the blue '''Analyze''' button in the top right corner to visualize your adjusted data.
*'''Export''' your data to the appropriate location.
[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]
[[ Category: Equipment Protocols ]]
*Click on the '''File Folder icon''' and find your folder (or the folder in which you saved your run).
*Select the file with extension '''.eds'''. The program will automatically take you to the '''Results''' tab of your run.
*Above the graph, use the dropdown box to select '''Melt Curve'''.
*Click on '''Target''' above the plate schematic to select one target at a time.
**Verify that there is only one (prominent) peak for each of your targets.
**Verify that each of your replicates amplified for each target.
**If there is a target that yields 2+ peaks over all samples, mark that target as unusable in the '''Primer Database'''.
**Exclude samples that did not the above criteria by clicking the well not the plate schematic and '''Omit''' under the '''Action''' dropdown box above the plate schematic.
*Check the technical replicates by choosing the '''Table View''' and arranging them according to target. Verify that the '''CT''' values have <1.5 difference. Omit outliers.
*Click '''Next''' and '''Export''' your results to the appropriate project folder.
*Under '''App Connect''' on the right hand side click on '''Relative Quantification''' (qPCR). Open your run.
*Under the '''Overview''' tab, assign biological groups to each of your samples (if needed).
*If you need to omit samples from the analysis, do so under the '''Data Review''' tab by clicking on the affected target, highlighting, and omitting the wells with outliers.
*Under the '''Analysis''' tab click the blue '''Analyze''' button in the top right corner to visualize your adjusted data.
*'''Export''' your data to the appropriate location.
[[ Category: Molecular Biology ]]
[[ Category: RNA ]]
[[ Category: qPCR ]]
[[ Category: Transcription ]]
[[ Category: Expression ]]
[[ Category: Equipment Protocols ]]
