ZipScript™ One-Step RT-qPCR Kit

Product Description

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. If a passive reference dye, such as ROX, is required for your assay, it can be purchased separately (P/N: Y9390).

Quality Control Analysis

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

Kit Contents:
25X ZipScript Enzyme Mix 2X ZipScript Reaction Buffer I

Notes:
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.

Protocol

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/probe mix. 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/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. Follow the table below to set up the reaction mix. It is recommended to make a master mix to minimize variations and potential errors.
   

   Components	Volume/Rxn	Final Conc
   Nuclease-Free Water	To a final 20 µL reaction volume	–
   2X ZipScript Reaction Buffer I	10 µL	11X
   50X ROX (optional)	0.4 µL	1X
   Primer/Probe Mix	X µL	Variable
   25X ZipScript Enzyme Mix	0.8 µL	0.8 µL
   RNA Template	Variable	–

6. Seal PCR plate and spin briefly to bring down reagents.

Thermal Cycling Conditions:

Program the cycling conditions based on the recommendations below.

Standard Cycling Program*

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