Instruction manual for the Stratagene StrataScript One-Tube RT-PCR System with Easy-A High-Fidelity PCR Cloning Enzyme, covering protocols and troubleshooting.
This document is an instruction manual for the Stratagene StrataScript One-Tube RT-PCR System with Easy-A High-Fidelity PCR Cloning Enzyme (Catalog #600168). The system combines cDNA synthesis, high-fidelity PCR amplification, and the generation of 3'-A overhangs in a single tube to detect RNA targets ranging from 0.1 to 6 kb.
| Easy-A HiFi PCR cloning enzyme quantity | 125 U |
|---|---|
| RT-PCR buffer concentration | 10× |
| dNTP mix concentration | 40 mM (10 mM of each dNTP) |
| Control mRNA concentration | 50 pg/ μl |
| Recommended RNA OD260/280 range | 1.8–2.0 |
| Recommended incubation temperature for cDNA synthesis | 42°C |
| Recommended incubation duration for cDNA synthesis | 30 minutes |
Both poly(A)+ and total RNA samples can be used as starting material.
Stratagene recommends a 42°C incubation for most targets.
Primers can be designed to anneal to sequences in exons on opposite sides of an intron, making the genomic product larger.