The One Step RT-PCR Master Mix Kit* allows rapid, sensitive analysis of gene expression from tissues and cells. One Step RT-PCR Master Mix Kit can replace methods for detecting and quantifying gene expression such as Northern blots, in situ hybridization, dot blots, S nuclease assays and conventional two step RT-PCR. The kit utilizes 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, optimized 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.

We recommend using 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. This kit is ideal for the rapid screening of gene expression. Each kit provides sufficient reagents to perform 50 RT-PCR analyses. Positive Control RNA and Control Primers are also included.

Features and Benefits:

• 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

• Ideal for gene expression studies

• Optimized buffer conditions and antibody-mediated hot start for increased sensitivity

• Rapid enzyme activation step (30 s) avoids damage of template RNA

Components:

• 2 x 625 µl 2X One Step RT-PCR Master Mix

• 1 x 200 µl 50 mM Mn(OAc)₂

• 1 x 1.1 ml RNase Free Water

• 1 x 50 µl Primer F (10 pmol/µl, 5'-TCC ACC ACC CTG TTG CTG TA-3')

• 1 x 50 µl Primer R (10 pmol/µl, 5'-ACC ACA GTC CAT GCC ATC AC-3')

• 1 x 50 µl Positive Control RNA (5 x 10⁸ copies/ml)

*Manufactured by Toyobo and distributed by EMD. Not available from Merck in Japan.

*Manufactured by Toyobo and distributed by EMD. Not available from Merck in Japan.