Aug 28, 2018

Public workspacePCR Protocol for TaqMan® Genotyping Assays

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.sfzebp6
  • 1Instituto Federal de Educação, Ciência e Tecnologia do Norte de Minas Gerais;
  • 2Instituto René Rachou - Fiocruz Minas;
  • 3Departamento de Enfermagem Materno-infantil e Saúde Pública, Escola de Enfermagem, Universidade Federal de Minas Gerais - UFMG
Eyleen Nabyla Alvarenga Niitsuma
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.sfzebp6
External link: https://doi.org/10.1371/journal.pone.0205234
Protocol Citation: Eyleen Nabyla Alvarenga Niitsuma, Gabriel da Rocha Fernandes, Francisco Carlos Félix Lana 2018. PCR Protocol for TaqMan® Genotyping Assays. protocols.io https://dx.doi.org/10.17504/protocols.io.sfzebp6
Manuscript citation:
Niitsuma ENA, Fernandes GdR, Lana FCF (2018) The TLR1 gene is associated with higher protection from leprosy in women. PLoS ONE 13(10): e0205234. doi: 10.1371/journal.pone.0205234
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: August 07, 2018
Last Modified: August 28, 2018
Protocol Integer ID: 14553
Guidelines
1 Keep all reagents protected from light until you are ready to use them. Excessive exposure to light may affect the fluorescent probes.
2 Minimize freeze-thaw cycles.
3 Prior to use:
– Mix the TaqMan Genotyping Master Mix thoroughly by swirling the bottle.
– Thaw any frozen TaqMan reagents by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly.
– Resuspend the TaqMan reagents by vortexing, then centrifuge the tube briefly.
– Thaw any frozen genomic DNA samples by placing them on ice. When thawed, resuspend the samples by vortexing, then centrifuge the tubes briefly.
Materials
MATERIALS
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
STEP MATERIALS
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
Protocol materials
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
Before start
1. Extract and purify genomic DNA.
2. Quantitate the gDNA in samples by spectophotometry.
Normalize the samples of  gDNA.
Note
The DNA concentration per sample is 20ng / μL.
Calculate the number of reactions to be performed for each assay, including recommended controls.
Note
Use negative and positive control. Prepare excess volume to account for pipetting errors.
Prepare the reaction mix - volume per well is 10.5 μL for a 96-well plate.
Prepare the reaction mix - volume per well is 10.5 μL for a 96-well plate.
Swirl the bottle of TaqMan® Genotyping Master Mix gently to mix the contents. Vortex and centrifuge the Genotyping Assay Working Stock, then mix briefly. Pipette the required volumes of TaqMan® Genotyping Master Mix and Genotyping Assay mix into a sterile tube. Cap the tube. 
Amount0.5 µL Genotyping Assay Working Stock (40x)
Amount10 µL TaqMan® Genotyping Master Mix (2x)
ReagentTaqMan® Genotyping Master MixApplied Biosystems (ThermoFisher Scientific)Catalog #4371355
ReagentTaqMan® SNP Genotyping AssaysApplied Biosystems (ThermoFisher Scientific)Catalog #4351376
Vortex the tube briefly to mix the components. Centrifuge the tube briefly to spin down the contents and to eliminate air bubbles from the solution.
Prepare the reaction plate
Prepare the reaction plate
Pipette 10.5 of the reaction mix (Master mix genotyping and TaqMan Assay) into each well of the reaction plate.
Into each well of the plate, pipette 2ul of normalized gDNA sample (concentration of 20ng/ul) and 7.5ul of ultrapure water.
Be sure to include wells for use as no template controls (no gDNA and 9.5ul of ultrapure water).
Note
Use a calibrated, positive-displacement pipettor to minimize contamination and error. Be sure that no cross-contamination occurs from well to well.
Cover the plate with MicroAmp® Optical Adhesive Film and seal the plate.
ReagentMicroAmp® Optical Adhesive FilmApplied Biosystems (ThermoFisher Scientific)Catalog #4360954
Centrifuge the plate briefly to spin down the contents and eliminate air bubbles from the solutions.
Perform the PCR
Perform the PCR
Place the plate in a Real-Time PCR instrument. Use the thermal cycling conditions specified.
Enzyme activation
Temp. 95°C - Duration 10 minutes - Cycles HOLD
Denaturation
Temp. 95°C - Duration 15 seconds - Cycles 40
Annealing/Extension
Temp. 60°C - Duration 1 minute - Cycles 40
PCR plate read and analysis
PCR plate read and analysis