Jul 23, 2020
qPCR Assay
This protocol is a draft, published without a DOI.
- 1University of California, San Diego
- George Lab @ UCSDTech. support email: olgeorge@ucsd.edu
Protocol Citation: Kokila Shankar, Olivier George 2020. qPCR Assay. protocols.io https://protocols.io/view/qpcr-assay-7whhpb6
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: Working
We use this protocol and it's working
Created: October 02, 2019
Last Modified: July 23, 2020
Protocol Integer ID: 28329
Abstract
This protocol is for running qPCR assays using previously generated cDNA. This protocol has been adapted from ThermoFisher (https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2Fcms_042179.pdf&title=UHJvdG9jb2w6IFBvd2VyIFNZQlIgR3JlZW4gUENSIE1hc3RlciBNaXggYW5kIFJULVBDUg==)
Guidelines
This protocol was used for quantifying nAChR mRNA levels in rat brain tissue. Some aspects of the protocol (primer concentration, PCR cycle steps) may need to be adjusted for other primers or template DNA.
Materials
MATERIALS
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
qPCR primers
Power SYBR™ Green PCR Master MixThermo FisherCatalog #4368577
PCR Tubes & Caps, RNase-free, 0.2 mL (8-strip format)Thermo FisherCatalog #AM12230
Hard Shell 384-well PlateBio-Rad LaboratoriesCatalog ##HSP3805
384-well Plate Sealing FlimBio-Rad LaboratoriesCatalog ##MSB1001
Before start
Ensure all primers have been tested to optimize concentration and reaction temperatures and efficiency has been calculated (if performing relative quantification analysis). If performing absolute quantification analysis, ensure standard cDNA is prepared. Thaw all reagents on ice.
Make cDNA and H2O master mix by combining 20 ng cDNA with enough H2O for a total of 4.5 µL , and multiplying these values by the number of wells needed. Samples should be run in at least duplicate, but preferably triplicate.
Note
Make sure to account for pipetting error and make slightly more master mix than needed (~5%)
Make SYBR and primer master mix by combining 5 µL SYBR, 0.25 µL 10 micromolar (µM) forward primer, and 0.25 µL 10 micromolar (µM) reverse primer, and multiplying these values by the amount of wells needed.
Note
Make sure to account for pipetting error and make slightly more master mix than needed (~5%)
Add 4.5 µL cDNA + H2O mix and 5.5 µL SYBR + primers mix to each well, for a total reaction volume of 10 µL . Mix master mixes frequently while pipetting, and make sure contents of each well are mixed properly.
Note
Having a template can be helpful to ensure everything gets pipetted correctly!
Seal plate thoroughly, spin down quickly (2400 rpm for 00:01:00 is enough), and run reaction at the following conditions:
- 95 °C for 00:10:00
- 95 °C for 00:00:15
- 59 °C for 00:01:00
- Repeat steps 2-3 39X
- Run melt curve
Reaction is run using
Equipment
CFX384 Touch
NAME
qPCR machine
TYPE
BioRad
BRAND
#1855484
SKU