Nov 14, 2022

Public workspaceGoga Lab RT-qPCR protocol: QuantStudio6 Machine V.3

This protocol is a draft, published without a DOI.
  • 1UCSF
Jeremy.williams
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Protocol Citation: Jeremy.williams 2022. Goga Lab RT-qPCR protocol: QuantStudio6 Machine. protocols.io https://protocols.io/view/goga-lab-rt-qpcr-protocol-quantstudio6-machine-ci95uh86Version created by Jeremy.williams
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 14, 2022
Last Modified: November 14, 2022
Protocol Integer ID: 72733
Abstract
Guidelines for preparing RT-qPCR samples for QuantStudio 6 located in HSW7 lab space.
Attachments
Icon for GogaLab-RTqPCR-Preparation.xlsx
GogaLab-RTqPCR-Prepa...
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Guidelines
*start with full tip boxes and use tips in coordination with your plate map, so you never get lost
*watch 1uL volumes in the pipet tip like a hawk - major source of variability
*cap and gently vortex your *mixture per primer set* every 12 replicates or so, sometimes things separate
Materials
PowerUp SYBR Green Master Mix
Your generated cDNA samples
100uM single primer stocks
Before start
*Thaw on ice (leave time!) and keep all reagents on ice through preparation. Prepare the plate on ice.

Prior to plate preparation:
Prior to plate preparation:
Dilute stock IDT primers to 100uM (see note in Guidelines).
Use 'GogaLab-RTqPCR-Preparation' Excel spreadsheet to input your sample number and calculate reagent volumes.

Prepare your plate:
Prepare your plate:
Mix *total reagent volumes required* (see spreadsheet, green) for DI and PowerUP
Mix your forward and reverse primer pairs together, to a final dilution of 10uM forward and 10uM reverse. For example, add 10uL each of forward and reverse primers to 80uL PCR-quality DI for 100uL final volume.
Mix *mixtures per primer set* (blue) volumes together.
Pipet 1uL diluted cDNA into respective qPCR well, aiming for the sidewall of each respective well.
Add 19uL *mixture per primer set* into each respective well.
Seal plate, and spin down 1000rpm for 1 minute. Use bacterial, not tissue culture, centrifuge.
Load plate into centrifuge and proceed using QuantStudio software suite.