qPCR Master Kit v3 - Product Specifications

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.

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.

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.