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DESCRIPTION

Tth DNA polymerase is a thermostable DNA polymerase derived from the thermophilic bacteria Thermus thermophilus (Tth) HB8.
The enzyme has a reverse transcriptase activity in addition to a 5’→3’ polymerase activity and a double strand specific 5’→ 3’ exonuclease activity in the presence of Mn2+ ions. Therefore, this enzyme enables "one-step RT-PCR" including the reverse transcription and PCR steps. Kits for one-step RT-PCR (Code No. PCR-311F) and real-time PCR (Code No. QRT-101, 201) using this enzyme are also available.

FEATURES

  • Exhibits reverse transcriptase activity in the presence of Mn2+ ions.
  • Effective for the amplification of GC-rich targets and crude samples.
  • Effective for reverse transcription of RNA with complicated secondary structure due to the reaction occurring at high temperature (i.e., 60ºC).

APPLICATIONS

  • PCR
  • RT-PCR
  • Primer extension

SOURCE

E. coli strain carrying the cloned Taq DNA polymerase gene from Thermus thermophilus (Tth) HB8.

UNIT DEFINITION

One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTP into an acid insoluble material in 30 min at 75ºC.

STORAGE CONDITION

10 mM Tris-HCl (pH 7.5), 300 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1% Tween® 20, 500 µg/ml BSA, 50% Glycerol.
Store at -20ºC

COMPONENTS

This reagent includes the following components for 100-200 reactions;

rTth DNA Polymerase (5U/µl)
10 x Buffer (Mg buffer)
Dilution buffer
2mM dNTPs

TYPICAL PCR REACTION SETUP

Normal PCR

Component Volume Final Concentration
10x Buffer 5 ml
2mM dNTPs 5 ml 0.2 mM each
10 pmol/ul Primer #1 1.0 ml 0.2 mM
10 pmol/ul Primer #2 1.0 ml 0.2 mM
Template DNA X ml Genomic DNA 10~1000 ng/50 µl
Plasmid DNA 1~50 ng/50 µl
cDNA ~200 ng (RNA equiv.)/50 µl
PCR grade water Y ml
Diluted rTth DNA polymerase (1.0U/µl) 1.25-2.5 ml 1.25-2.5 U / 50 µl
Total reaction volume 50 ml

PCR CYCLE CONDITIONS

*Extension time should be set at 1 min per 1 kb of target le

APPLICATION DATA

Example 1.Amplification of 180 bp–1.3 kb genes from human genomic DNA.

Distinct and specific amplified bands from 180 bp to 1.3 kb were observed with rTth DNA polymerase by 1% agarose gel electrophoresis.

Example 2.Amplification of the yeast actin and human 18s rRNA by RT-PCR

The single-enzyme RT-PCR with rTth DNA polymerase gave distinct amplification bands, whereas RT-PCR with M-MLV reverse transcriptase and rTaq DNA polymerase gave very faint bands.

REFERENCES

  • T.W. Myers, D.H. Gelfand, Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry, 30: 7661-7666 (1991).
  • K. Yamada, M. Terashima, M. Shimoyama, M. Tsuchiya, Arginine-specific ADP-ribosyltransferase on the surface of gizzard smooth muscle cells and the involvement of phosphatidylinositol 3-kinase in maintaining the activity of this transferase. J Biochem. 130: 335-40 (2001)