The One Step RT-PCR Master Mix Kit allows rapid, sensitive analysis of gene expression from tissues and cells. It can replace methods for detecting and quantifying gene expression such as Northern blots, in situ hybridisation, dot blots, S nuclease assays and conventional two step RT-PCR. The kit utilises recombinant Thermus thermophilus (rTth) DNA Polymerase, which acts as both a thermostable RNA dependent DNA polymerase and a DNA dependent DNA polymerase. The rTth DNA Polymerase is provided in a 2X master mix with an antibody for antibody-mediated hot start, optimised buffer, and ultrapure deoxynucleotides. Antibody-mediated Hot Start enhances specificity of both reverse transcription and PCR. The kit enables cDNA synthesis from input RNA followed by PCR amplification of the cDNA in a single reaction, with no additional hands-on requirement for buffer changes or adding reagents. Typically, detection of a specific transcript requires only 2 hours.
- Ideal for gene expression studies
- Robust one-step, one-enzyme master mix system for easy reaction assembly
- Eliminates the risk of cross contamination associated with two-step RT-PCR protocols
- High-temperature (60 °C) for reverse transcription enhances read-through of RNA secondary structure
- Optimized buffer conditions and antibody-mediated hot start for increased sensitivity
- Rapid enzyme activation step (30 s) avoids damage of template RNA
NOTE: It is recommended to use either two gene-specific primers or oligo (dT) and one gene-specific 5'-primer with the kit. Although rTth adds 3' dA overhangs, it is generally not recommended for PCR product cloning because the rTth error rate is higher than standard Taq DNA polymerase.
Lieferumfang: Kit includes 2×625 µl 2X One Step RT-PCR Master Mix, 1×200 µl 50 mM Mn(OAc)2, 1×1,1 ml RNase-free water, 1×50 µl Primer F (10 pmol/µl, 5'-TCC ACC ACC CTG TTG CTG TA-3'), 1×50 µl primer R (10 pmol/µl, 5'-ACC ACA GTC CAT GCC ATC AC-3'), 1×50 µl positive control RNA (5×108 copies/ml).
