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DESCRIPTION

RT-PCR Quick Master Mix provides a 2 x Master Mix for RT-PCR using a thermostable DNA polymerase derived from Thermus thermophilus (Tth) HB8 (1). Tth DNA polymerase exhibits reverse transcriptase activity in addition to DNA polymerase activity in the presence of Mn2+ ions. Therefore, this system enables "one-step RT-PCR" including reverse transcription and PCR steps. This kit is suitable for high-throughput RT-PCR, and decreases contamination risks.

FEATURES

  • This kit enables one-step RT-PCR including reverse transcription and PCR steps. This kit is suitable for high-throughput RT-PCR and decreases contamination risks.
  • Compared with normal reverse transcriptases from retroviruses (e.g., M-MLV reverse transcriptase), Tth DNA polymerase can work at a higher temperature. Therefore, this kit has the advantage of amplifying targets with complex conformations and GC-rich content.
  • Hot-start technology using anti-Tth DNA polymerase antibodies enables highly efficient and specific amplification.

APPLICATIONS

  • One-step RT-PCR

STORAGE CONDITION

Store at -20ºC

COMPONENTS

2 x RT-PCR Quick Master Mix 625 µl x 2
50 mM Mn(OAc)2 200 µl
Nuclease-free water 1100 µl
Positive control RNA
(human G3PDH mRNA, 5 x 105 copies/µl) 50 µl
Control Primer F (10 pmol/µl) 50 µl
Control Primer R (10 pmol/µl) 50 µl

APPLICATION DATA

Example 1.Comparison of kit sensitivity

The target gene (human G3PDH: 450 bp) was amplified using in vitro translated G3PDH mRNA with one-step RT-PCR kits. As shown in Fig.1, the RT-PCR Master Mix [Code No. PCR-311] exhibited greater sensitivity than the other commercial kit.

Fig. 1

Amplification of the 450-bp human G3PDH gene

Example 2.Detection of cdc2 mRNA using one-step RT-PCR

The human cdc2 gene (900 bp) was amplified using mRNA (5 ng and 0.5 ng) purified from a human cell line. As shown in Fig. 2, target genes were successfully amplified using the RT-PCR Quick Master Mix.

Fig. 2

Amplification of the 900-bp human cdc gene

REFERENCES

  • Myers T. W. and Gelfand D. H. , Biochemistry, 30: 7661-6 (1991)