Total bacterial quantification was done by quantitative real-time PCR of 16s ribosomal small subunit (ssu16s) DNA as target. Custom Taqman assay for ssu16s rDNA target was developed using 5′-AAACTCAAATGAATTGACGGGG-3′ as forward, 5′-TCGTTGCGGGACTTAACCC-3′ as reverse, and 6FAM-ACGCGAAGAACCTTAC as probe. Ten-microliter reactions were prepared using 3 μL of DNA (concentrations of DNA adjusted to ~20 ng/μl), 5 μL TaqMan 2X Environmental Master Mix, 0.5 μL 20X primer-probe mix, and 1.5 μL nuclease-free H2O (all reagents from Applied Biosystems, Inc.). Amplifications were performed for 40 cycles in a 7500 Fast Real-Time PCR System (Applied Biosystems, Inc.). Copy numbers for each transcript in each sample were calculated using 7500 Fast Real-Time PCR System Sequence Detection Software v. 1.3.1 (Applied Biosystems, Inc.) against a known copy number standard curve. A standard curve was generated using serial 10-fold dilutions of known copies of a plasmid DNA as copy number standards.
Primer probe mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 20X primer-probe mix is a ready-to-use solution containing the necessary components for real-time PCR assays. It includes forward and reverse primers, as well as a fluorescently labeled probe, all in a single, concentrated format. This product is designed to simplify the setup process and ensure consistent performance across multiple reactions.
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Lab products found in correlation
2 protocols using primer probe mix
1
Quantitative Real-Time PCR for Bacterial Enumeration
2
Quantitative Real-Time PCR Analysis
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Quantitative real-time PCR (qPCR) was performed using inventoried Taqman assays from Applied Biosystems, CA, USA) (20x Primer Probe mix) corresponding to EWSR1 (Assay ID Hs03024497_ft), ACTB (Assay ID Hs01060665_g1) and GAPDH (Assay ID Hs02758991_g1). XBP1. All PCR reactions were performed with TaqMan Fast Advanced Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus Real Time PCR System in accordance with the standard protocols. The amount of each target gene relative to the housekeeping gene 18S, ACTB and GAPDH was determined using the comparative threshold cycle (Ct) method. For each sample, the relative amount of each target gene was calibrated against a control sh-RNA transfected cell line (Cont). All assays were performed in triplicate.
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