One-Step RT-PCR Kit, 100 Reactions - Must be stored at - 20° C
abm’s One-Step RT-PCR Kit contains all necessary reagents for both reverse transcription and
PCR amplification to occur in a single reaction tube. Specifically, this One-Step RT-PCR kit
contains EasyScriptTM RTase and BestaqTM DNA Polymerase in a convenient format for highly
sensitive and specific RT-PCR using any RNA template. Our proprietary RT-PCR buffer contains
stabilizers and enhancers that optimize the two reactions in a “single step”. Together with a
specially formulated RT-PCR buffer, this One-Step RT-PCR kit offers the end-users an efficient,
easy to use and reliable alternative to conventional “two-step” sequential RT-PCR.
Storage Conditions Store all components at -20°C in a non-frost-free freezer. All components are stable for 1 year
from the date of shipping when stored and handled properly.
Protocol RT-PCR should be assembled in a nuclease-free environment. RNA sample preparation,
reaction mixture assembly, PCR, and subsequent reaction analysis should be performed
in separate areas. The use of “clean”, automatic pipettors designated for PCR and
aerosol-resistant barrier tips are recommended.
1. Prepare the following reaction mixture in a qPCR tube on ice:
| Components | Volume | Concentration |
| Total RNA or poly(A) + mRNA | Variable | 1 ng - 2 µg/rxn
1 pg - 2 ng/rxn |
| 2X One-Step RT-PCR Buffer | 25 µl | 1X |
| EasyScript™ RTase (200 U/µl) | 1 µl | 20o U/rxn |
| Bestaq™ DNA POlymerase (5 U/µl) | 2 µl | 10 U/rxn |
| Forward Primer (10 µM) | 2.5 µl | 500 nM |
| Reverse Primer (10 µM) | 2.5 µl | 500 nM |
| Nuclease-free H2O | Up to 50 µl | - |
Note: Gene-specific primers should be used. 2. Gently mix and ensure all the components are at the bottom of the amplification tube.
Centrifuge briefly if needed.
3. Program the thermal cycler so that cDNA synthesis is followed immediately by PCR
amplification automatically. The following cycling conditions were established using a
DNA Thermal Cycler 2400 (Perkin Elmer) and may have to be altered for other thermal
cyclers.
| Steps | Temperature | Duration | Cycle(s) |
| cDNA Synthesis | 42° C | 30 mins | 1 |
| Initial Denaturation | 94° C | 3 mins | 1 |
| Denaturation | 94° C | 30 secs |
30 - 35
|
| Annealing | 55° C | 30 secs |
| Extension | 72° C | 1 kb/min |
| Final Extension | 72° C | 5 mins | 1 |
| Holding | 4° C | - | 1 |
Note:
1) The thermal cycling program listed above is optimized for primers with an annealing
temperature at 55°C.
2) An optional touchdown thermal cycling program can also be used to replace the steps after
the initial cDNA synthesis in the table above.
4. Analyze the amplification products by agarose gel electrophoresis and visualize the
nucleic acids via ethidium bromide or SafeViewTM staining (abm Cat. No. G108). Use
appropriate molecular weight standards.
