TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Upstream tips
Amplicon size should be consistent for each target sequence and limited to approximately 65 - 100 bp.

Publication protocol

RNA isolation was performed with the NucleoSpin RNA II kit (Macherey‐Nagel), as described by the manufacturer. Using cDNAs of transfected HEK293 cells, we performed quantitative expression analysis with the 7900HT Fast Real‐Time PCR System (Applied Biosystems). The PCR amplification was performed with TaqMan gene expression assay Hs01026070_m1 for human TRPM4. For control, we studied expression of a cotransfected human CD8 gene with TaqMan gene expression assay Hs00233520_m1. For each sample, triplicate determinations were performed in a 96‐well optical plate using 80 ng of cDNA, 1 μL of 20× TaqMan Gene Expression Assay, 10 μL of 2× TaqMan Fast Universal PCR Master Mix No AmpErase UNG, and 5 μL of water in each 20 μL reaction. Plates were heated for 20 seconds at 95°C, and then 40 cycles of 1 second at 95°C and 20 seconds at 60°C were applied. Relative TRPM4 expression (ΔCt) was calculated by subtracting the signal threshold cycle (Ct) of the control (CD8) from the Ct value of TRPM4. Subsequently, ΔΔCt values were obtained for each mutant by subtracting the ΔCt of the TRPM4 WT from the ΔCt of each mutant. Results were then linearized by calculating 2−expΔΔCt.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing PCR Multiplex PCR - Mammalian DNA using TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing PCR Multiplex PCR - Mammalian DNA using TaqMan™ Fast Universal PCR Master Mix (2X), no AmpErase™ UNG from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.