MBiotech¡¯s One-step 2X RT-PCR Master Premix is a mixture of ExAct Taq DNA polymerase, M-MLV reverse transcriptase, dNTPs, reaction buffer, and stabilizer. Users can perform RT-PCR by simply adding total RNA or poly(A) RNA as substrate and specific primer pair to the premix. One-step 2X RT-PCR Master Premix also contains glycerol and bromophenol blue as a tracking dye, enabling a direct analysis of PCR products on an analytic gel electrophoresis. Stabilizer in premix enables prolonging a shelf life at -20¡É. You do not need to dissolve the mixture prior to RT-PCR reaction as does competitor¡¯ freeze-dried one. The composition is optimized to reduce non-specific amplification and to minimize primer-dimer formation.

One-step RT-PCR.

Performance tests.

 

Store at -20¡É for one year.

 

 

One-step RT-PCR from Cultured Human Cells

 

1. GAPDH
2. PAI-1
3. erbB2
4. b4 integrin
5. p21Waf1

 

PCR Preparation (20 ul total volume)
100 ng total RNA isolated from human dermal fibroblast
10 ul 2X RT-PCR premix
0.5 ul each primer (10 pmol/ul)

 

PCR Cycle Condition

42¡É 20 min
95¡É 5 min

  94¡É 30 sec
  58¡É 30 sec        x 35 cycles
  72¡É 45 sec

72¡É 5 min

 

One-step RT-PCR of RNA Viral Genome from Infected Animal

PCR Preparation (20 ul total volume)
100 ng total RNA isolated from viral infected pig blood
10 ul 2X RT-PCR premix
0.5 ul each primer (10 pmol/ul)

 

PCR Cycle Condition

42¡É 20 min
95¡É 5 min

  94¡É 30 sec
  58¡É 30 sec        x 35 cycles
  72¡É 45 sec

72¡É 5 min