---
title: qPCR Master Kit v3 - Product Specifications
description: PathoSEEK qPCR Master Kit v3 Product Specifications and Minimum Release Criteria
---
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# qPCR Master Kit v3 - Product Specifications
## PathoSEEK® qPCR Master Kit v3 provides the core reagents required to run PathoSEEK® qPCR assays on a real-time qPCR platform. Relative to prior kit versions, v3 includes an increased quantity of enzyme in the qPCR Master Mix to accommodate the growing number of multiplexed presence/absence assays supported on the PathoSEEK® platform.
### Kit Components:
The PathoSEEK® qPCR Master Kit v3 (Product SKU 420201) includes the following components:
- Reaction Buffer (10x)
- qPCR Master Mix (5x)
- Nuclease-Free Water
### Component Specifications / Minimum Release Criteria
Each lot of the components below is tested against the following minimum release criteria prior to release.
**3.1 qPCR Master Mix (5x)**
| **Test / Parameter** | **Minimum Release Criteria** |
| --- | --- |
| **Functional Testing (qPCR)** | Master Mix is functionally tested in qPCR with human cDNA template, resulting in a standard curve with a calculated qPCR efficiency of 90–110%, and a dynamic range of 5 orders of magnitude. |
| **Non-Specific DNase Activity (16 hour, Master Mix)** | A 50 µl reaction in qPCR Master Mix containing 1 µg of T3 DNA, and a separate reaction containing Lambda-HindIII DNA, incubated for 16 hours at 37ºC, results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. |
| **qPCR DNA Contamination (E. coli Genomic)** | A minimum of 1 µl of qPCR Master Mix is screened for the presence of E. coli genomic DNA using SYBR® Green qPCR with primers specific for the E. coli 16S rRNA locus, quantified against a standard curve generated from purified E. coli genomic DNA. Measured contamination is ≤ 1 E. coli genome. |
| **RNase Activity Assay (4 Hour Digestion)** | A 10 µl reaction in Reaction Buffer containing 40 ng of a 300-base single-stranded RNA and a minimum of 1 µl of qPCR Master Mix is incubated at 37ºC for 4 hours. \>90% of the substrate RNA remains intact as determined by gel electrophoresis using fluorescent detection. |
**3.2 Reaction Buffer (10x)**
| **Test / Parameter** | **Minimum Release Criteria** |
| --- | --- |
| **Conductivity** | The conductivity of Reaction Buffer is between 36 and 54 mS at 25°C. |
| **pH** | The pH of Reaction Buffer is between pH 7.8 and 8.0 at 25°C. |
| **Endonuclease Activity (Nicking)** | A 50 µl reaction in Reaction Buffer containing 1 µg of supercoiled PhiX174 DNA, incubated for 4 hours at 37°C, results in \<10% conversion to the nicked form as determined by agarose gel electrophoresis. |
| **Non-Specific DNase Activity (16 hour)** | A 50 µl reaction in Reaction Buffer containing 1 µg of PhiX174-HaeIII DNA, incubated for 16 hours at 37ºC, results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. |
| **Functional Testing – Restriction Digest (MscI)** | A 50 µl reaction in Reaction Buffer plus 100 µg/ml Bovine Serum Albumin containing 1 µg of Lambda DNA and 1 unit of MscI, incubated for 1 hour at 37ºC, results in complete digestion of the substrate DNA as determined by agarose gel electrophoresis. |
| **Functional Testing – Restriction Digest (ClaI)** | A 50 µl reaction in Reaction Buffer plus 100 µg/ml Bovine Serum Albumin containing 1 µg of Lambda dam- DNA and 1 unit of ClaI, incubated for 1 hour at 37ºC, results in complete digestion of the substrate DNA as determined by agarose gel electrophoresis. |
| **RNase Activity** | A 10 µl reaction in Reaction Buffer containing 40 ng of a 300-base single-stranded RNA is incubated at 37ºC for 16 hours. \>90% of the substrate RNA remains intact as determined by fluorescent detection. |
**3.3 Nuclease-Free Water**
| **Test / Parameter** | **Minimum Release Criteria** |
| --- | --- |
| **UV-Visible Scan** | A UV-Visible scan using a spectrophotometer covering the range of 200 nm to 800 nm results in no detectable peaks above background. |
| **Endotoxin Testing (Endosafe®)** | Each test channel of the cartridge is loaded with 25 µl of Nuclease-free Water and placed into the Endosafe MCS reader for analysis, resulting in a measurement of \<0.01 EU/ml. |
| **Endonuclease Activity (Nicking)** | A 50 µl reaction in rCutSmart™ Buffer containing 1 µg of supercoiled PhiX174 DNA with Nuclease-free Water, incubated for 4 hours at 37ºC, results in \<10% conversion to the nicked form as determined by agarose gel electrophoresis. |
| **Non-Specific DNase Activity (16 hour)** | A 50 µl reaction in rCutSmart™ Buffer containing 1 µg of PhiX174-HaeIII DNA with Nuclease-free Water, incubated for 16 hours at 37ºC, results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. |
| **qPCR DNA Contamination (E. coli Genomic)** | Nuclease-free Water is used to make a qPCR master mix and screened across a 96-well plate for the presence of E. coli genomic DNA using 40 cycles of SYBR® Green qPCR with primers specific for the E. coli 16S rRNA locus. Melt curve analysis results in \<5% positive samples above background. |
| **RNase Activity (Extended Digestion)** | A 10 µl reaction in Reaction Buffer containing 40 ng of RNA transcript with Nuclease-free Water is incubated at 37ºC for 16 hours. No detectable degradation of the RNA is observed by gel electrophoresis using fluorescent detection. |
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