ZipScript One-Step RT-qPCR Kit

• Highly sensitive and reproducible RT-qPCR solution
• Automation friendly
• Features a blend of EnzScript and Phoenix Hot Start DNA Polymerase
• Efficient RNA dependent multiplex qPCR formulation

The ZipScript One-Step RT-qPCR Kit is a highly sensitive and reproducible RT-qPCR solution optimized for real-time PCR. The 25X enzyme mix is accompanied by a 2X reaction buffer.

Inquire for lyophilized or lyo ready options.

Mix properties

• Storage temperature: –25°C to –15°C

The functionality of the ZipScript One-Step RT-qPCR Kit is evaluated by amplification of two mRNA transcripts in a one-step quantitative RT-qPCR assay. The amplification threshold (C_q) of the test lot is compared to a reference lot.

ZipScript One-Step RT-qPCR reaction setup

1. All components and reaction set up should be kept on ice.
2. Thaw the 2X ZipScript Reaction Buffer I completely and vortex for 3–5 seconds to mix thoroughly. Quick spin to collect contents if necessary.
3. Prepare primer and probe mixture. A final concentration of 0.4–0.9 µM for each primer and 0.1–0.5 µM for probe is recommended. However, the optimal concentration for primers and probe needs to be empirically determined for each assay.
4. Determine the number of reactions to prepare, including No Template Controls (NTCs). Add 10% extra volume to compensate for the pipetting loss.
5. Use the amounts listed in the table below to set up the reaction mix. We recommend making a master mix to minimize variations and potential errors.

   Components	Volume per reaction	Final concentration
   Nuclease-free water	To a final reaction volume of 20 µl	–
   2X ZipScript Reaction Buffer I	10 µl	11X
   50X ROX (optional)	0.4 µl	1X
   Primer and probe mixture	Variable	Variable
   25X ZipScript Enzyme Mix	0.8 µl	0.8 µl
   RNA Template	Variable	Variable
6. Seal PCR plate and spin briefly to bring down reagents.
7. Program the cycling conditions based on the recommendations below.

   Steps	Temperature	Time	Cycles
   Reverse transcription	50°C	15 minutes	1
   Taq activation and initial denaturation	95°C	2 minutes	1
   Denaturation	95°C	15 seconds	40
   Annealing and extension*	60°C	30–60 seconds	-

   * Cycling parameters can be modified (especially annealing and extension parameters) to fit specific primer and probe selection.

   Quality control analysis

   The functionality of the ZipScript is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (C_q) of the test lot is compared to a reference lot.

   Enzyme components were tested prior to formulation of the master mix and found free of contaminating endonucleases and exonucleases. Enzyme purity was >99% as determined by SDS-PAGE and negligible E.coli genomic DNA contamination was confirmed by qPCR. Specific activity was verified for each enzyme pre-formulation.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• One-step RT-qPCR