Home Protocols One-Step RT-PCR Protocols (E5315)

One-Step RT-PCR Protocols (E5315)

Quick-Load OneTaq One-Step Reaction Mix can be used if direct gel loading is desired.

Standard Protocol

Thaw system components and mix by inverting several times.

  1. Mix the following components, except RNA, in sterile RNase-free microfuge tubes.
    OneTaq One-Step Reaction Mix (2X)   25 μl
    OneTaq One-Step Enzyme Mix (25X)   2 μl
    Gene-specific Forward Primer (10 μM)   2 μl
    Gene-specific Reverse Primer (10 μM)   2 μl
    Nuclease-free H2O   19–x μl
    Total RNA (up to 1 μg)   x μl
    ------------------------------------------------------------------------------------------   
    Total volume   50 μl

  2. Add RNA template last, and start reactions immediately, as follows:
    CYCLE STEP TEMPERATURE TIME CYCLES
    Reverse Transcription 48°C 15–30 minutes 1
    Initial Denaturation 94°C 1 minute 1
    Denaturation
    Annealing
    Extension    		 94°C
    50–65°C
    68°C    		 15 seconds
    30 seconds
    1 minute per kb    		 40
    Final Extension 68°C 5 minutes 1
    Hold 4°C ∞ 1

Alternative Protocol:

If denaturation of template RNA is desired, use the following protocol:

  1. Mix RNA sample and primers in sterile, RNase-free microfuge tubes.
    Total RNA (up to 1 μg)   x μl
    Gene-specific reverse primer (10 μM)   2 μl
    OneTaq One-Step Reaction Mix (2X)   25 μl
    Nuclease-free H2O   19–x µl
    ------------------------------------------------------------------------------------------ 
    Total Volume   46 µl

    Denature for 5 minutes at 65°C. Put promptly on ice.

  2. Add the following components to the tube:
    OneTaq One-Step Enzyme Mix (25X)   2 μl
    Gene-specific Forward Primer (10 μM)   2 μl

  3. Add tubes to the thermocycler, and run the following program:
    CYCLE STEP TEMPERATURE TIME CYCLES
    Reverse Transcription 48°C 15–30 minutes 1
    Initial Denaturation 94°C 1 minute 1
    Denaturation
    Annealing
    Extension    		 94°C
    50–65°C
    68°C    		 15 seconds
    30 seconds
    1 minute per kb    		 40
    Final Extension 68°C 5 minutes 1
    Hold 4°C ∞ 1

No-RT Negative Control Reaction:

Mix the following components in a sterile RNase-free microfuge tube:

OneTaq One-Step Reaction Mix (2X)   25 μl
Gene-specific Forward Primer (10 μM)   2 μl
Gene-specific Reverse Primer (10 μM   )2 μl
Hot Start OneTaq DNA polymerase (not provided)   2 U
Nuclease-free H2O   19–x μl
Total RNA (up to 1 μg)   x μl
------------------------------------------------------------------------------------------ 
Total volume   50 μl

Proceed with reaction conditions as follows:
CYCLE STEP TEMPERATURE TIME CYCLES
Reverse Transcription 48°C 15–30 minutes 1
Initial Denaturation 94°C 1 minute 1
Denaturation
Annealing
Extension		 94°C
50–65°C
68°C		 15 seconds
30 seconds
1 minute per kb		 40
Final Extension 68°C 5 minutes 1
Hold 4°C ∞ 1